Anti-cd20 antibodies and fusion proteins therof and methods of use

ABSTRACT

The present invention provides humanized, chimeric and human anti-CD20 antibodies and CD20 antibody fusion proteins that bind to a human B cell marker, referred to as CD20, which is useful for the treatment and diagnosis of B-cell disorders, such as B-cell malignancies and autoimmune diseases, and methods of treatment and diagnosis.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. patent application Ser. No.12/212,359, filed Sep. 17, 2008, which is a continuation of U.S. patentapplication Ser. No. 11/534,103, filed Sep. 21, 2006, now U.S. Pat. No.7,435,803, which is a continuation of U.S. patent application Ser. No.10/366,709, filed Feb. 14, 2003, now U.S. Pat. No. 7,151,164, whichclaims priority to U.S. Provisional Application No. 60/356,132, filedFeb. 14, 2002 and U.S. Provisional Application No. 60/416,232, filedOct. 7, 2002.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to humanized, chimeric and human anti-CD20antibodies, particularly monoclonal antibodies (mAbs) therapeutic anddiagnostic conjugates of humanized, chimeric and human anti-CD20antibodies and methods of treating B cell lymphomas and leukemias andvarious autoimmune diseases using humanized, chimeric and humananti-CD20 antibodies. The present invention relates to antibody fusionproteins or fragments thereof comprising at least two anti-CD20 mAbs orfragments thereof or at least one anti-CD20 MAb or fragment thereof andat least one second MAb or fragment thereof, other than the antiCD20 MAbor fragment thereof. The humanized, chimeric and human anti-CD20 mAbs,fragments thereof, antibody fusion proteins thereof or fragments thereofmay be administered alone, as a therapeutic conjugate or in combinationwith a therapeutic immunoconjugate, with other naked antibodies, or withtherapeutic agents or as a diagnostic conjugate. The present inventionrelates to DNA sequences encoding humanized, chimeric and humananti-CD20 antibodies, and antibody fusion proteins, vectors and hostcells containing the DNA sequences, and methods of making the humanized,chimeric and human anti-CD20 antibodies.

2. Background

The immune system of vertebrates consists of a number of organs and celltypes which have evolved to accurately recognize foreign antigens,specifically bind to, and eliminate/destroy such foreign antigens.Lymphocytes, amongst others, are critical to the immune system.Lymphocytes are divided into two major sub-populations, T cells and Bcells. Although inter-dependent, T cells are largely responsible forcell-mediated immunity and B cells are largely responsible for antibodyproduction (humoral immunity).

In humans, each B cell can produce an enormous number of antibodymolecules. Such antibody production typically ceases (or substantiallydecreases) when a foreign antigen has been neutralized. Occasionally,however, proliferation of a particular B cell will continue unabated andmay result in a cancer known as a B cell lymphoma. B-cell lymphomas,such as the B-cell subtype of non-Hodgkin's lymphoma, are significantcontributors to cancer mortality. The response of B-cell malignancies tovarious forms of treatment is mixed. For example, in cases in whichadequate clinical staging of non-Hodgkin's lymphoma is possible, fieldradiation therapy can provide satisfactory treatment. Still, aboutone-half of the patients die from the disease. Devesa et al., J. Nat'lCancer Inst. 79:701 (1987).

The majority of chronic lymphocytic leukemias are of B-cell lineage.Freedman, Hematol. Oncol. Clin. North Am. 4:405 (1990). This type ofB-cell malignancy is the most common leukemia in the Western world.Goodman et al., Leukemia and Lymphoma 22:1 (1996). The natural historyof chronic lymphocytic leukemia falls into several phases. In the earlyphase, chronic lymphocytic leukemia is an indolent disease,characterized by the accumulation of small maturefunctionally-incompetent malignant B-cells having a lengthened lifespan. Eventually, the doubling time of the malignant B-cells decreasesand patients become increasingly symptomatic. While treatment canprovide symptomatic relief, the overall survival of the patients is onlyminimally affected. The late stages of chronic lymphocytic leukemia arecharacterized by significant anemia and/or thrombocytopenia. At thispoint, the median survival is less than two years. Foon et al., AnnalsInt. Medicine 113:525 (1990). Due to the very low rate of cellularproliferation, chronic lymphocytic leukemia is resistant to cytotoxicdrug treatment.

Traditional methods of treating B-cell malignancies, includingchemotherapy and radiotherapy, have limited utility due to toxic sideeffects. The use of monoclonal antibodies to direct radionuclides,toxins, or other therapeutic agents offers the possibility that suchagents can be delivered selectively to tumor sites, thus limitingtoxicity to normal tissues. Also, the presence of B-cell antigens onthese B-cell malignancies makes them optimal targets for therapy withunconjugated B-cell antibodies, such as against CD19, CD20, CD21, CD23,and CD22 markers on B-cells. HLA-DR and other antigens may serve astargets for normal and malignant B-cells, although they are alsoexpressed on other cell types. Further, certain MUC1, MUC2, MUC3, andMUC4 antigens, preferably MUC1, are also expressed in differenthematopoietic malignancies, including B-cell tumors expressing CD20 andother B-cell markers. Still other antigen targets, such as thoseassociated with the vascular endothelium of tumors, including tenascin,vascular endothelium growth factor (VEGF), and placental growth factor(PlGF), as well as other categories of antigens associated with B-cellmalignancies, such as oncogene products, are also suitable targets forsaid complementary antibodies for use in the present invention.

B cells comprise cell surface proteins which can be utilized as markersfor differentiation and identification. One such human B-cell marker isthe human B lymphocyte-restricted differentiation antigen Bp35, referredto as CD20. CD20 is expressed during early pre-B cell development andremains until plasma cell differentiation. CD20 is expressed on bothnormal B cells and malignant B cells whose abnormal growth can lead toB-cell lymphomas. Antibodies against the CD20 antigen have beeninvestigated for the therapy of B-cell lymphomas. For example, achimeric anti-CD20 antibody, designated as “IDEC-C2B8,” has activityagainst B-cell lymphomas when provided as unconjugated antibodies atrepeated injections of doses exceeding 500 mg per injection. Maloney etal., Blood 84:2457 (1994); Longo, Curr. Opin. Oncol. 8:353 (1996). About50 percent of non-Hodgkin's patients, having the low-grade indolentform, treated with this regimen showed responses. Therapeutic responseshave also been obtained using ¹³¹]-labeled B1 anti-CD20 murinemonoclonal antibody when provided as repeated doses exceeding 600 mg perinjection. Kaminski et al., N. Engl. J. Med. 329:459 (1993); Press etal., N. Engl. J. Med. 329:1219 (1993); Press et al., Lancet 346:336(1995). However, these antibodies, whether provided as unconjugatedforms or radiolabeled forms, have not shown high rates of objective anddurable responses in patients with the more prevalent and lethal form ofB-cell lymphoma, the intermediate or aggressive type. Therefore, a needexists to develop an immunotherapy for B-cell malignancies that achievesa therapeutic response of significant duration.

Additional studies targeting CD20 surface antigen have been demonstratedusing an anti-CD20 murine monoclonal antibody, IF5, which wasadministered by continuous intravenous infusion to B cell lymphomapatients. Extremely high levels (>2 grams) of 1F5 were reportedlyrequired to deplete circulating tumor cells, and the results weredescribed as being “transient.” Press et al., “Monoclonal Antibody 1F5(Anti-CD20) Serotherapy of Human B-Cell Lymphomas.” Blood 69/2:584-591(1987). However, a potential problem with this approach is thatnon-human monoclonal antibodies (e.g., murine monoclonal antibodies)typically lack human effector functionality, i.e., they are unable tomediate complement-dependent lysis or lyse human target cells throughantibody-dependent cellular toxicity or Fc-receptor mediatedphagocytosis. Furthermore, non-human monoclonal antibodies can berecognized by the human host as a foreign protein and, therefore,repeated injections of such foreign antibodies can lead to the inductionof immune responses leading to harmful hypersensitivity reactions. Formurine-based monoclonal antibodies, this is often referred to as a HumanAnti-Mouse Antibody (HAMA) response.

The use of chimeric antibodies is more preferred because they do notelicit as strong a HAMA response as murine antibodies. Chimericantibodies are antibodies which comprise portions from two or moredifferent species. For example, Liu, A. Y. et al, “Production of aMouse-Human Chimeric Monoclonal Antibody to CD20 with PotentFc-Dependent Biologic Activity” J. Immun. 139/10:3521-3526 (1987),describe a mouse/human chimeric antibody directed against the CD20antigen. See also, PCT Publication No. WO 88/04936. However, noinformation is provided as to the ability, efficacy or practicality ofusing such chimeric antibodies for the treatment of B cell disorders inthe reference. It is noted that in vitro functional assays (e.g.,complement-dependent lysis (CDC); antibody dependent cellularcytotoxicity (ADCC), etc.) cannot inherently predict the in vivocapability of a chimeric antibody to destroy or deplete target cellsexpressing the specific antigen. See, for example, Robinson, R. D. etal., “Chimeric mouse-human anti-carcinoma antibodies that mediatedifferent anti-tumor cell biological activities,” Hum. Antibod.Hybridomas 2:84-93 (1991) (chimeric mouse-human antibody havingundetectable ADCC activity). Therefore, the potential therapeuticefficacy of a chimeric antibody can only truly be assessed by in vivoexperimentation, preferably in the species of interest for the specifictherapy.

One approach that has improved the ability of murine monoclonalantibodies to be effective in the treatment of B-cell disorders has beento conjugate a radioactive label or chemotherapeutic agent to theantibody, such that the label or agent is localized at the tumor site.For example, the above-referenced 1F5 antibody and other B-cellantibodies have been labeled with ¹³¹I and were reportedly evaluated forbiodistribution in two patients. See Eary, J. F. et al., “Imaging andTreatment of B-Cell Lymphoma” J. Nuc. Med. 31/8:1257-1268 (1990); seealso, Press, O. W. et al., “Treatment of Refractory Non-Hodgkin'sLymphoma with Radiolabeled MB-1 (Anti-CD37) Antibody” J. Clin. One.7/8:1027-1038 (1989) (indication that one patient treated with¹³¹I-labeled IF-5 achieved a partial response); Goldenberg, D. M. etal., “Targeting, Dosimetry and Radioimmunotherapy of B-Cell Lymphomaswith ¹³¹I-Labeled LL2 Monoclonal Antibody” J. Clin. Oncol. 9/4:548-564(1991) (three of eight patients receiving multiple injections reportedto have developed a HAMA response to this CD22 murine antibody);Appelbaum, F. R. “Radiolabeled Monoclonal Antibodies in the Treatment ofNon-Hodgkin's Lymphoma” Hem./Oncol. Clinics of N. A. 5/5:1013-1025(1991) (review article); Press, O. W. et al. “Radiolabeled-AntibodyTherapy of B-Cell Lymphoma with Autologous Bone Marrow Support.” NewEngland Journal of Medicine 329/17: 1219-12223 (1993) (¹³¹I-labeledanti-CD20 antibody IF5 and B1); and Kaminski, M. G. et al“Radioimmunotherapy of B-Cell Lymphoma with [¹³¹I] Anti-B1 (Anti-CD20)Antibody”. NEJM 329/7:459 (1993) (¹³¹I-labeled anti-CD20 antibody B1;hereinafter “Kaminski”); PCT published application WO 92/07466(antibodies conjugated to chemotherapeutic agents such as doxorubicin ormitomycin). However, these approaches have not eliminated the obstaclesassociated with using murine antibodies, despite the fact that manypatients with lymphoma who have received prior aggressive cytotoxicchemotherapy are immune suppressed, thus having lower HAMA rates thanlymphoma patients who have not been heavily pretreated.

Autoimmune diseases are a class of diseases associated with B-celldisorders. Examples include immune-mediated thrombocytopenias, such asacute idiopathic thrombocytopenic purpura and chronic idiopathicthrombocytopenic purpura, myasthenia gravis, lupus nephritis, lupuserythematosus, and rheumatoid arthritis. The most common treatments arecorticosteroids and cytotoxic drugs, which can be very toxic. Thesedrugs also suppress the entire immune system, can result in seriousinfection, and have adverse affects on the bone marrow, liver andkidneys. Other therapeutics that have been used to treat Class IIIautoimmune diseases to date have been directed against T-cells andmacrophages. There is a need for more effective methods of treatingautoimmune diseases, particularly Class III autoimmune diseases.

To address the many issues related to B-cell disorders and theirtreatment, the present invention provides humanized, chimeric and humananti-CD20 monoclonal antibodies with the same complementaritydetermining regions (CDRs) that bind to the CD20 antigen of the presentinvention used alone, conjugated to a therapeutic agent or incombination with other treatment modalities, for the treatment of B celllymphomas and leukemias and autoimmune disorders in humans and othermammals without the adverse responses associated with using murineantibodies.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides humanized, chimeric andhuman anti-CD20 antibodies that bind to a human B cell marker, referredto as CD20, which is useful for the treatment and diagnosis of B-celldisorders, such as B-cell malignancies and autoimmune diseases.

The present invention further provides methods of treatment of mammaliansubjects, such as humans or domestic animals, with one or morehumanized, chimeric and human CD20 antibodies, alone, as an antibodyfusion protein, as a therapeutic conjugate alone or as part of anantibody fusion protein, in combination, or as a multimodal therapy,with other antibodies, other therapeutic agents or immunomodulators oras an immunoconjugate linked to at least one therapeutic agent,therapeutic radionuclide or immunomodulator. These humanized, chimericand human CD20 antibodies can also be used as a diagnostic imaging agentalone, in combination with other diagnostic imaging agents, and/or inconjunction with therapeutic applications.

The present invention additionally is directed to anti-CD20 mAbs orfragments thereof that contain specific murine CDRs or a combination ofmurine CDRs from more than one murine or chimeric anti-CD20 MAb thathave specificity for CD20. These mAbs can be humanized, chimeric orhuman anti-CD20 mAbs. The present invention is further directed to lightand/or heavy chain variable regions or fragments thereof of theseanti-CD20 Mabs and to light and/or heavy chains or fragments thereofthat have specficity for CD20.

The present invention is also directed to antibody fusion proteinscomprising at least two anti-CD20 mAbs or fragments thereof or a firstMAb comprising an anti-CD20 mAbs or fragments thereof and a second MAb.

The present invention is further directed to a therapeutic or diagnosticconjugates of the anti-CD20 mAbs or fragments thereof or antibody fusionproteins of the anti-CD20 mAbs or other mAbs or fragments thereof boundto at least one therapeutic agent or at least one diagnostic agent.Antibody fusion proteins with multiple therapeutic agents of the same ordifferent type are encompassed by the present invention.

The present invention is additionally directed to a method of using theanti-CD20 mAbs or fragments thereof or antibody fusion proteins thereofor fragments thereof for therapy, either alone, in combination with eachother, as the antibody component of a therapeutic immunoconjugate withone or more therapeutic agents or each administered in combination withone or more therapeutic agents or with an immunoconjugate with one ormore therapeutic agents.

The present invention further is directed to a method of using theanti-CD20 mAbs or fragments thereof or antibody fusion proteins thereofor fragments thereof as a diagnostic bound to one or more diagnosticagents.

The present invention additionally is directed to a method ofpretargeting a cell in a patients suffering from a B-cell lymphoma orleukemia or an autoimmune disease using an antibody fusion protein orfragment thereof of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 discloses the V gene sequences cloned by RT-PCR from a hybridomacell line producing a murine anti-CD20, and the deduced amino acidsequences of the variable light (FIG. 1A) (SEQ ID NOS: 32 & 33) andheavy chain (FIG. 1B) (SEQ ID NOS: 34 & 35) of the A20 antibody,designated as A20Vk and A20VH, respectively. Underlined arrows indicatethe sequences of the PCR primers used for cloning. The putative CDRregion sequences, as defined by the Kabat numbering scheme, are shown inbold and underlined Amino acid sequences are given as single-lettercodes below the corresponding nucleotide sequence. The Kabat numberingscheme was used for amino acid residues Amino acid residues numbered bya letter represent the insertion residue according to Kabat, and havethe same number as that of the previous residue. For example, residues82, 82A, 82B and 82C in FIG. 1B are indicated as 82 A, B, and C,respectively.

FIG. 2 discloses the Vk, the variable light chain, and the VH, thevariable heavy chain, sequences of cA20, a chimeric anti-CD20 antibody.The CDR region sequences are shown in bold and underlined. The aminoacid residues and the nucleotides are numbered sequentially and samenumbering system is used for humanized V sequences. The light chainvariable region is shown in FIG. 2A (SEQ ID NOS 36 & 37) and the heavychain variable region is shown in FIG. 2B (SEQ ID NOS: 38 & 39). Thenumbering system is the same as for FIG. 1. The restriction sites usedfor constructing cA20 are underlined.

FIG. 3 shows a comparison of the binding affinities of the chimeric A20(cA20), and murine A20, (A20), in a cell surface competitive bindingassay against ¹²⁵I-labled A20. Increasing concentrations of cA20 blockedthe binding of radiolabeled A20 to Raji cells (as depicted by closedcircles) in a comparable manner as that of murine A20 (depicted byclosed diamonds).

FIG. 4 compares the amino acid sequences of the variable heavy chain(VH) and variable light chain (Vk) of human antibodies, and chimeric andhumanized anti-CD20 antibodies. FIG. 4A compares the amino acidsequences of the variable heavy chain (VH) of the human antibodies, EU(SEQ ID NO: 40) and NEWM (SEQ ID NO: 43) (FR4 only), the chimericantibody, (cA20VH) (SEQ ID NO: 39) and two humanized antibodies,(hA20VH1 (SEQ ID NO: 41) and hA20VH2 (SEQ ID NO: 42)) and FIG. 4Bcompares the amino acid sequences of the variable light chain (Vk) ofthe human antibody, (REIVk) (SEQ ID NO: 44), a chimeric antibody,(cA20Vk) (SEQ ID NO: 37), and a humanized antibody, (hA20Vk) (residues20-125 of SEQ ID NO: 46). Dots indicate that the residues in A20 areidentical to the corresponding residue in the human antibody. The CDRsare identified as a boxed region. The Kabat numbering scheme was used tonumber the amino acid residues.

FIG. 5 discloses the nucleotide sequences of hA20 light chain V genes,(hA20Vk) (FIG. 5A) (SEQ ID NOS: 45 & 46), and heavy chain V genes,hA20VH1 (FIG. 5B) (SEQ ID NOS: 47 & 48) and hA20VH2 (FIG. 5C) (SEQ IDNOS: 49 & 50), as well as the adjacent flanking sequences of the VKpBR2(FIG. 5A) and VHpBS2 (FIGS. 5B and 5C) staging vectors, respectively.The non-translated nucleotide sequences are shown in lowercase. Therestriction sites used for subcloning are underlined and indicated. Thesecretion signal peptide sequence is indicated by a double underline.Numbering of Vk and VH amino acid residues is same as that in FIG. 2.

FIG. 6 shows the results of a cell surface competitive binding assay tocompare the binding activity of two humanized A20 antibodies, (hA20-1and hA20-2), with that of A20, cA20 and a chimeric anti-CD20 MAb, c2B8.FIG. 6A shows hA20-1 (closed triangles) and hA20-2 (closed circles) andthe murine anti-CD20 antibody, A20 (closed squares) competed equallywell for the binding of ¹²⁵I-A20 to Raji cells. FIG. 6B shows hA20-1(closed circles), cA20 (closed squares) and c2B8 (closed diamonds)competed equally well for the binding of ¹²⁵I-c2B8 to Raji cells.

FIG. 7 discloses the constant region of a human IgG1 (CH-hinge) (FIG.7A) (SEQ ID NOS: 51 & 52) and the constant region of a human kappa chain(Ck) (FIG. 7B) (SEQ ID NOS: 53 & 54).

FIG. 8 is a competitive cell surface binding assay. Ag-bindingspecificity and affinity studies of humanized anti-CD20 Abs (cA20, hA20,and c1F5, purified by affinity chromatography on a Protein A column)were evaluated by a cell surface competitive binding assay with murine2B8 and rituximab (IDEC Pharmaceuticals Corp., San Diego, Calif.). FIG.8 (A) is a comparison of the binding activities of cA20 (square), hA20-1(triangle) and hA20-1 (circle) with that of m2B8 (diamond); FIG. 8 (B)compares of the binding activities of cA20 (square), c1F5 (triangle) andrituximab (diamond).

FIG. 9 is a study comparing the binding activities of hA20 with otheranti-CD20 Abs, including rituximab and murine B1, by a cell surfacecompetitive binding assay. A constant amount (100,000 cpm, ˜10 ìCi/ìg)of ¹²⁵I-labeled rituximab was incubated with Raji cells in the presenceof varying concentrations (0.2-700 nM) of competing Abs, hA20(triangle), mB1 (Downward triangle) or rituximab (square) at 4° C. for1-2 h.

FIG. 10 depicts the cytotoxic effect of crosslinked hA20 and other CD20Abs on cultured lymphoma cells. Total cell and viable cell populationswere measured by (A) trypan blue staining and cell counting or (B) MTTassay.

FIG. 11 is a graph of in vivo therapy studies with various anti-CD20 andother Abs. Raji cells administered i.v. to SCID mice, to create a Rajilymphoma model of disseminated disease.

FIG. 12 is a graph depicting in vivo therapy with hA20 and hLL2. Rajicells administered i.v. to SCID mice, to create a Raji lymphoma model ofdisseminated disease.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

As discussed above, anti-CD20 antibodies that are unconjugated orlabeled with a therapeutic radionuclide, have failed to provide highrates of objective and lasting responses in patients with intermediateor aggressive forms of B-cell lymphoma. The present invention provides ahumanized, a chimeric and a human anti-CD20 antibody, and antibodyfusion proteins thereof, useful for treatment of mammalian subjects,humans and domestic animals, alone, as a conjugate or administered incombination with other therapeutic agents, including other nakedantibodies and antibody therapeutic conjugates.

The anti-CD20 mAbs of the present invention contain specific murine CDRsor a combination of murine CDRs from more than one murine or chimericanti-CD20 MAb that have specificity for the CD20 antigen. The anti-CD20mAbs of the present invention are humanized, chimeric or human mAbs,light and/or heavy chains thereof or light and/or heavy chain variableregions thereof, and they contain the amino acids of the CDRs of amurine anti-CD20 MAb and retain substantially the B-cell and B-celllymphoma and leukemia cell targeting of the murine anti-CD20 MAb. TheCDRs of the light chain variable region of the anti-CD20 MAb comprisesCDR1 comprising amino acids RASSSVSYIH (SEQ ID NO: 1), RASSSLSFMH (SEQID NO: 2) or RASSSVSYMH (SEQ ID NO: 3) CDR2 comprising amino acidsATSNLAS (SEQ ID NO: 4) and CDR3 comprising amino acids QQWTSNPPT (SEQ IDNO: 5), HQWSSNPLT (SEQ ID NO: 6) or QQSFSNPPT (SEQ ID NO: 7) and theCDRs of the heavy chain variable region of the anti-CD20 MAb comprisesCDR1 comprising amino acids SYNMH (SEQ ID NO: 8) CDR2 comprising aminoacids AIYPGNGDTSYNQKFKG (SEQ ID NO: 9) and CDR3 comprising amino acidsSTYYGGDWYFDV (SEQ ID NO: 10), STYYGGDWYFNV (SEQ ID NO: 11),SHYGSNYVDYFDV (SEQ ID NO: 12) or VVYYSNSYWYFDV (SEQ ID NO: 13). Thehumanized antibody further comprises the framework regions of the lightand heavy chain constant regions of a human antibody.

In one embodiment, the humanized and chimeric MAb or fragment thereofdoes not contain the CDR3 of the heavy chain variable region comprisingSTYYGGDWYFNV (SEQ ID NO: 11). More preferably, CDR1 of the light chainvariable region does not comprise RASSSLSFMH (SEQ ID NO: 2) when theCDR3 of the light chain variable region comprises HQWSSNPLT (SEQ ID NO:6) and the CDR3 of the heavy chain variable region comprisesSHYGSNYVDYFDV (SEQ ID NO: 12). In another embodiment, the CDR3 of thelight chain variable region does not comprise HQWSSNPLT (SEQ ID NO: 6)when CDR1 of the light chain variable region comprises RASSSLSFMH (SEQID NO: 2) and when CDR3 of the heavy chain variable region comprisesSHYGSNYVDYFDV (SEQ ID NO: 12). In a further embodiment, the CDR3 of theheavy chain variable region does not comprise SHYGSNYVDYFDV (SEQ ID NO:12) when the CDR1 of the light chain variable region comprisesRASSSLSFMH (SEQ ID NO: 2) and the CDR3 of the light chain variableregion comprises HQWSSNPLT (SEQ ID NO: 6). In another embodiment, theCDR1 of the light chain variable region does not comprise RASSSVSYMH(SEQ ID NO: 3) when the CDR3 of the light chain variable regioncomprises QQSFSNPPT (SEQ ID NO: 7) and the CDR3 of the heavy chainvariable region comprises VVYYSNSYWYFDV (SEQ ID NO:13).

Further, in another embodiment, the anti-CD20 monoclonal antibody (MAb)or fragment thereof does not contain CDR3 of the light chain variableregion of amino acids QQSFSNPPT (SEQ ID NO: 7) when CDR1 of the lightchain variable region comprises RASSSVSYMH (SEQ ID NO: 3) and the CDR3of the heavy chain variable region comprises VVYYSNSYWYFDV (SEQ ID NO:13). Additionally, the anti-CD20 MAb does not contain CDR3 of the heavychain variable region with amino acids VVYYSNSYWYFDV (SEQ ID NO: 13)when the CDR1 of the light chain variable region comprises RASSSVSYMH(SEQ ID NO: 3) and the CDR3 of the light chain variable region comprisesQQSFSNPPT (SEQ ID NO: 7).

In a preferred embodiment, the humanized anti-CD20 (hCD20) monoclonalantibody or antigen-binding fragment thereof comprising thecomplementarity determining regions (CDRs) of at least one murineanti-CD20 MAb variable region and the framework regions (FRs) of atleast one human MAb variable region, wherein said humanized anti-CD20MAb or fragment thereof retains substantially the B-cell and B-celllymphoma and leukemia cell targeting of said murine anti-CD20 MAb. Thehumanized antibody's variable region may comprise a light chain variableregion, a heavy chain variable region or a both light and heavy chainvariable regions. The humanized antibody or fragment thereof may furthercomprise light and heavy chain constant regions of at least one humanantibody.

The humanized anti-CD20 MAb or fragment thereof of the present inventioncomprises the CDRs of a murine anti-CD20 MAb and the framework (FR)regions of the light and heavy chain variable regions of a humanantibody, while retaining substantially the B-cell, and B-cell lymphomaand leukemia cell targeting of the parent murine antiCD20 MAb, andwherein the CDRs of the light chain variable region of the murineantiCD20 MAb comprises CDR1 comprising amino acids RASSSVSYIH (SEQ IDNO: 1), CDR2 comprising amino acids ATSNLAS (SEQ ID NO: 4) and CDR3comprising QQWTSNPPT (SEQ ID NO: 5) and the CDRs of the heavy chainvariable region of murine anti-CD20 MAb comprises CDR1 comprising aminoacids SYNMH (SEQ ID NO: 8), CDR2 comprising amino acidsAIYPGNGDTSYNQKFKG (SEQ ID NO: 9) and CDR3 comprising amino acidsSTYYGGDWYFDV (SEQ ID NO: 10). But the humanized anti-CD20 MAb orfragment thereof may further contain in the FRs of the light and heavychain variable regions of the antibody at least one amino acid from thecorresponding FRs of the murine MAb. The humanized MAbs may furthercontain the light and heavy chain constant regions of a human antibody.Specifically, the humanized anti-CD20 MAb or fragment thereof containsat least one amino acid residue 1, 5, 27, 30, 38, 48, 67, 68, 70, 95,115 and 116 of the murine heavy chain variable region of FIG. 4A,designated as hA20VH1 or hA20VH2 and of at least one amino acid residue4, 21, 35, 38, 45, 46, 59, 99, 104 and 106 of the murine light chainvariable region FIG. 4B, designated hA20Vk. One or more of the murineamino acid sequences can be maintained in the human FR regions of thelight and heavy variable chains if necessary to maintain proper bindingor to enhance binding to the CD20 antigen. More preferably the humanizedantiCD20 MAb or fragment thereof of the present invention comprises thehA20Vk of FIG. 4B and the hA2VH1 of FIG. 4A. Most preferably, thehumanized anti-CD20 MAb or fragment thereof of the present inventioncomprises the hA20Vk of FIG. 4B and the hA20VH2 of FIG. 4A. This lattersequence contains more human amino acid sequences in the FRs of the VH2chain than the VH1, and thus is more humanized.

The preferred chimeric anti-CD20 (cCD20) MAb or fragment thereof of thepresent invention comprises the CDRs of a murine anti-CD20 MAb and theFR regions of the light and heavy chain variable regions of the murineanti-CD 20 MAb, i.e., the Fvs of the parental murine MAb, and the lightand heavy chain constant regions of a human antibody, wherein thechimeric anti-CD20 MAb or fragment thereof retains substantially theB-cell, and B-cell lymphoma and leukemia cell targeting of the murineanti-CD20 MAb, wherein the CDRs of the light chain variable region ofthe chimeric anti-CD20 MAb comprise CDR1 comprising amino acidsRASSSVSYIH (SEQ ID NO: 1), RASSSLSFMH (SEQ ID NO: 2) or RASSSVSYMH (SEQID NO: 3) CDR2 comprising amino acids ATSNLAS (SEQ ID NO: 4) and CDR3comprising amino acids QQWTSNPPT (SEQ ID NO: 5), HQWSSNPLT (SEQ ID NO:6) or QQSFSNPPT (SEQ ID NO: 7) and the CDRs of the heavy chain variableregion of the chimeric anti-CD20 MAb comprise CDR1 comprising aminoacids SYNMH (SEQ ID NO: 8) CDR2 comprising amino acids AIYPGNGDTSYNQKFKG(SEQ ID NO: 9) and CDR3 comprising STYYGGDWYFDV (SEQ ID NO: 10),STYYGGDWYFNV (SEQ ID NO: 11), SHYGSNYVDYFDV (SEQ ID NO: 12) orVVYYSNSYWYFDV (SEQ ID NO: 13) with the following provisos,

(a) wherein the CDR3 of the heavy chain variable region does notcomprise STYYGGDWYFNV (SEQ ID NO: 11), when the CDR1 of the light chainvariable region comprises amino acids RASSSVSYIH (SEQ ID NO: 1), CDR2 ofthe light chain variable region comprises amino acids ATSNLAS (SEQ IDNO: 4), CDR3 of the light chain variable region comprises amino acidsQQWTSNPPT (SEQ ID NO: 5), CDR1 of the heavy chain variable regioncomprises amino acids SYNMH (SEQ ID NO: 8), and CDR2 of the light chainvariable region comprises amino acids AIYPGNGDTSYNQKFKG (SEQ ID NO: 9)

(b) wherein the CDR3 of the heavy chain variable region does notcomprise SHYGSNYVDYFDV (SEQ ID NO: 12), when the CDR1 of the light chainvariable region comprises amino acids RASSSLSFMH (SEQ ID NO: 2), CDR2 ofthe light chain variable region comprises amino acids ATSNLAS (SEQ IDNO: 4), CDR3 of the light chain variable region comprises amino acidsHQWSSNPLT (SEQ ID NO: 6), CDR1 of the heavy chain variable regioncomprises amino acids SYNMH (SEQ ID NO: 8), and CDR2 of the light chainvariable region comprises amino acids AIYPGNGDTSYNQKFKG (SEQ ID NO: 9)and

(c) wherein the CDR3 of the heavy chain variable region does notcomprise VVYYSNSYWYFDV (SEQ ID NO: 13), when the CDR1 of the light chainvariable region comprises amino acids RASSSVSYMH (SEQ ID NO: 3), CDR2 ofthe light chain variable region comprises amino acids ATSNLAS (SEQ IDNO: 4), CDR3 of the light chain variable region comprises amino acidsQQSFSNPPT (SEQ ID NO: 7), CDR1 of the heavy chain variable regioncomprises amino acids SYNMH (SEQ ID NO: 8), and CDR2 of the light chainvariable region comprises amino acids AIYPGNGDTSYNQKFKG (SEQ ID NO: 9).

More preferably the chimeric anti-CD20 MAb or fragment thereofcomprising the complementarity-determining regions (CDRs) of a murineanti-CD20 MAb and the framework (FR) regions of the light and heavychain variable regions of the murine anti-CD20 MAb and further, thelight and heavy chain constant regions of a human antibody, wherein thechimeric anti-CD20 MAb or fragment thereof retains substantially theB-cell, and B-cell lymphoma and leukemia cell targeting of the murineanti-CD20 MAb, wherein the CDRs of the light chain variable region ofthe chimeric anti-CD20 MAb comprises the CDRs shown in FIGS. 4B and 4A,respectively, designated cA20Vk and cA20VH. Most preferably, thechimeric anti-CD20 MAb or fragment thereof comprises the light and heavychain variable regions of murine anti-CD20 MAb shown in FIGS. 4B and 4A,respectively, designated cA20Vk and cA20 VH.

The present invention also encompasses a human anti-CD20 MAb or fragmentthereof comprising the light and heavy chain variable, wherein saidhuman CD20 MAb retains substantially the B-cell, and B-cell lymphoma andleukemia cell targeting and cell binding characteristics of a murineanti-CD20 MAb, wherein the CDRs of the light chain variable region ofthe human anti-CD20 MAb comprises the same CDRs as set forth above forthe chimeric and humanized anti-CD20 mAbs and as shown in FIGS. 4A and4B. This human anti-CD20 MAb or fragment thereof further comprises lightand heavy chain constant regions of at least one human antibody.

The present invention is also intended to encompass antibody fusionproteins or fragments thereof comprising at least two anti-CD20 mAbs orfragments thereof, as described above. The antibody fusion protein orfragment thereof of the present invention is also intended to encompassan antibody fusion protein or fragment thereof comprising at least onefirst anti-CD20 MAb or fragment thereof as described above and at leastone second MAb or fragment thereof, other than the antiCD20 MAb orfragment described above. More preferably this second MAb is a MAbreactive with CD4, CD5, CD8, CD14, CD15, CD19, CD21, CD22, CD23, CD25,CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, B7,MUC1, MUC2, MUC3, MUC4, Ia, HM1.24, HLA-DR, tenascin, VEGF, PlGF, anoncogene, oncogene product, or a combination thereof, and even ananti-CD20 MAb that is different than the anti-CD20 MAb described herein.The antibody fusion proteins of the present invention may be composed ofone CD20 MAb and one or more of the second mAbs to provide specificityto different antigens, and are described in more detail below.

The humanized, chimeric and human anti-CD20 antibody may possessenhanced affinity binding with the epitope, as well as antitumor andanti-B-cell activity, as a result of CDR mutation and manipulation ofthe CDR and other sequences in the variable region to obtain a superiortherapeutic agent for the treatment of B-cell disorders, includingB-cell lymphomas and leukemias and autoimmune diseases. Modification tothe binding specificity, affinity or avidity of an antibody is known anddescribed in WO 98/44001, as affinity maturation, and this applicationsummarizes methods of modification and is incorporated in its entiretyby reference.

It may also be desirable to modify the antibodies of the presentinvention to improve effector function, e.g., so as to enhanceantigen-dependent cell-mediated cytotoxicity (ADCC) and/or complementdependent cytotoxicity (CDC) of the antagonist. One or more amino acidsubstitutions or the introduction of cysteine in the Fc region may bemade, thereby improving internalization capability and/or increasedcomplement-mediated cell killing and ADCC. See Caron et al., J. Ex. Med.176:1191-1195 (1991) and Shopes, B. J. Immunol. 148:2918-2022 (1992),incorporated herein by reference in their entirety. An antibody fusionprotein may be prepared that has dual Fc regions with both enhancedcomplement lysis and ADCC capabilities.

The present invention is also directed to DNA sequences comprising anucleic acid encoding a MAb or fragment thereof selected from the groupconsisting

(a) an anti-CD20 MAb or fragment thereof as described herein,

(b) an antibody fusion protein or fragment thereof comprising at leasttwo of the anti-CD20 mAbs or fragments thereof,

(c) an antibody fusion protein or fragment thereof comprising at leastone first MAb or fragment thereof comprising the anti-CD20 MAb orfragment thereof as described herein and at least one second MAb orfragment thereof, other than the antiCD20 MAb or fragment thereof, and

(d) an antibody fusion protein or fragment thereof comprising at leastone first MAb or fragment thereof comprising the anti-CD20 MAb orfragment thereof and at least one second MAb or fragment thereof,wherein the second MAb is a MAb reactive with CD4, CD5, CD8, CD14, CD15,CD19, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52,CD54, CD74, CD80, CD126, B7, MUC1, MUC2, MUC3, MUC4, Ia, HM1.24, HLA-DR,tenascin, VEGF, PlGF, an oncogene, oncogene product, or a combinationthereof.

Also encompassed by the present invention are expression vectorscomprising the DNA sequences. These vectors contain the light and heavychain constant regions and the hinge region of the human immunoglobulin,in the case of vectors for use in preparing the humanized, chimeric andhuman anti-CD20 mAbs or antibody fusion proteins thereof or fragmentsthereof. These vectors additionally contain, where required, promotersthat express the mAbs in the selected host cell, immunoglobulin enhancesand signal or leader sequences. Vectors that are particularly useful inthe present invention are pdHL2 or GS, particularly when used to expressa chimeric, humanized or human antibodies, such as gigs, where thevector codes for the heavy and light chain constant regions and hingeregion of IgG1. More preferably, the light and heavy chain constantregions and hinge region are from a human EU myeloma immunoglobulin,where optionally at least one of the amino acid in the allotypepositions is changed to that found in a different IgG1 allotype, andwherein optionally amino acid 253 of the heavy chain of EU based on theEU number system may be replaced with alanine. See Edelman et al., Proc.Natl. Acad. Sci. USA 63: 78-85 (1969), incorporated herein in itsentirety by reference.

Host cells containing the DNA sequences encoding the anti-CD20 mAbs orfragments thereof or antibody fusion proteins or fragments thereof ofthe present invention or host cells containing the vectors that containthese DNA sequences are encompassed by the present invention.Particularly useful host cells are mammalian cells, more specificallylymphocytic cells, such as myeloma cells, discussed in more detailbelow.

Also encompassed by the present invention is the method of expressingthe anti-CD20 MAb or fragment thereof or antibody fusion protein orfragment thereof comprising: (a) transfecting a mammalian cell with aDNA sequence of encoding the anti-CD20 mAbs or fragments thereof orantibody fusion proteins or fragments thereof, and (b) culturing thecell transfected with the DNA sequence that secretes the anti-CD20 orfragment thereof or antibody fusion protein or fragment thereof. Knowntechniques may be used that include a selection marker on the vector sothat host cells that express the mAbs and the marker can be easilyselected.

The present invention particularly encompasses B-lymphoma cell andleukemia cell targeting diagnostic or therapeutic conjugates comprisingan antibody component comprising an anti-CD20 MAb or fragment thereof oran antibody fusion protein or fragment thereof of the present inventionthat binds to the B-lymphoma or leukemia cell, that is bound to at leastone diagnostic or at least one therapeutic agent.

The diagnostic conjugate comprises the antibody component comprising ananti-CD20 MAb or fragment thereof or an antibody fusion protein orfragment thereof, wherein the diagnostic agent comprises at least onephotoactive diagnostic agent, and more preferably wherein the label is aradioactive label with an energy between 60 and 4,000 keV or anon-radioactive label. The radioactive label is preferably a gamma-,beta-, and positron-emitting isotope and is selected from the groupconsisting of ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁸⁶Y, ¹⁸⁶Re, ¹⁸⁸Re, ⁶²Cu, ⁶⁴Cu,¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ¹⁸F, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br andcombinations thereof.

The diagnostic conjugate of the present invention also utilizes adiagnostic agent, such as a contrast agent, for example, such asmanganese, iron or gadolinium.

The therapeutic conjugate of the present invention comprises an antibodycomponent comprising an antibody fusion protein or fragment thereof,wherein each of said mAbs or fragments thereof are bound to at least onetherapeutic agent. The therapeutic conjugate of preferably is selectedfrom the group consisting of a radioactive label, an immunomodulator, ahormone, a photoactive therapeutic agent, a cytotoxic agent, which maybe a drug or a toxin, and a combination thereof. The drugs useful in thepresent invention are those drugs that possess the pharmaceuticalproperty selected from the group consisting of antimitotic, antikinase,alkylating, antimetabolite, antibiotic, alkaloid, antiangiogenic,apoptotic agents and combinations thereof. More specifically, thesedrugs are selected from the group consisting of nitrogen mustards,ethylenimine derivatives, alkyl sulfonates, nitrosoureas, triazenes,folic acid analogs, COX-2 inhibitors, pyrimidine analogs, purineanalogs, antibiotics, enzymes, epipodophyllotoxins, platinumcoordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, antagonists,endostatin, taxols, camptothecins, anthracyclines, taxanes, and theiranalogs, and a combination thereof. The toxins encompassed by thepresent invention are selected from the group consisting of ricin,abrin, alpha toxin, saporin, ribonuclease (RNase), e.g., onconase, DNaseI, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

Useful therapeutic conjugates of the present invention areimmunomodulators selected from the group consisting of a cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof. Specifically useful arelymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors,such as interleukin (IL), colony stimulating factor, such asgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF)), interferon, such asinterferons-α, -β or -γ, and stem cell growth factor, such as designated“S1 factor”. More specifically, immunomodulator, such as IL-1, IL-2,IL-3, IL-6, IL-10, IL-12, IL-18, IL-21 interferon-γ, TNF-α or acombination thereof are useful in the present invention.

Particularly useful therapeutic conjugates comprise one or moreradioactive labels that have an energy between 60 and 700 keV. Suchradioactive labels are selected from the group consisting of ²²⁵Ac,⁶⁷Ga, ⁹⁰Y, ¹¹¹In, ¹³¹I, ¹²⁵I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ³²P, ⁶⁴Cu, ⁶⁷Cu,²¹²Bi, ²¹³Bi, ²¹¹At and combinations thereof. Other useful therapeuticconjugates are photoactive therapeutic agent, such as a chromogen ordye.

Other useful therapeutic conjugates comprise oligonucleotides,especially antisense oligonucleotides that preferably are directedagainst oncogenes and oncogene products of B-cell malignancies, such asbcl-2.

The present invention particularly encompasses methods of treating aB-cell lymphoma or leukemia cell disease or an autoimmune disease in asubject, such as a mammal, including humans, domestic or companion pets,such as dogs and cats, comprising administering to the subject atherapeutically effective amount of an anti-CD20 MAb or a fragmentthereof of the present invention, formulated in a pharmaceuticallyacceptable vehicle. This therapy utilizes a “naked antibody” that doesnot have a therapeutic agent bound to it. The administration of the“naked anti-CD20 antibody” can be supplemented by administering to thesubject concurrently or sequentially a therapeutically effective amountof another “naked antibody” that binds to or is reactive with anotherantigen on the surface of the target cell or that has other functions,such as effector functions in the Fc portion of the MAb, that istherapeutic and which is discussed herein. Preferred additional mAbs areat least one humanized, chimeric, human or murine (in the case ofnon-human animals) MAb selected from the group consisting of a MAbreactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23,CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80,CD126, B7, MUC1, Ia, HM1.24, and HLA-DR, tenascin, VEGF, PlGF, anoncogene, oncogene product, or a combination thereof, formulated in apharmaceutically acceptable vehicle.

Both the naked anti-CD20 therapy alone or in combination with othernaked mAbs as discussed above can be further supplemented with theadministration, either concurrently or sequentially, of atherapeutically effective amount of at least one therapeutic agent,formulated in a pharmaceutically acceptable vehicle. As discussed hereinthe therapeutic agent may comprises a cytotoxic agent, a radioactivelabel, an oligonucleotide, an immunomodulator, a hormone, an enzyme, anoligonucleotide, a photoactive therapeutic agent or a combinationthereof, formulated in a pharmaceutically acceptable vehicle.

In another therapeutic method, both the naked anti-CD20 therapy alone orin combination with other naked mAbs, as discussed above, can be furthersupplemented with the administration, either concurrently orsequentially, of a therapeutically effective amount of at least onetherapeutic conjugate, described herein and formulated in apharmaceutically acceptable vehicle. The antibody component of thetherapeutic conjugate comprises at least one humanized, chimeric, humanor murine (for non-human subjects) MAb selected from the groupconsisting of a MAb reactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20,CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54,CD74, CD80, CD126, B7, MUC1, MUC2, MUC3, MUC4, Ia, HM1.24, and HLA-DR,tenascin, VEGF, PlGF, an oncogene, oncogene product, or a combinationthereof, formulated in a pharmaceutically acceptable vehicle. Asdiscussed herein the therapeutic agent may comprise a cytotoxic agent, aradioactive label, an immunomodulator, a hormone, a photoactivetherapeutic agent or a combination thereof, formulated in apharmaceutically acceptable vehicle.

As described herein the present invention particularly encompasses amethod of treating a B-cell lymphoma or leukemia or an autoimmunedisease in a subject comprising administering to a subject atherapeutically effective amount of an antibody fusion protein orfragment thereof comprising at least two anti-CD20 mAbs or fragmentsthereof of the present invention or comprising at least one anti-CD20MAb or fragment thereof of the present invention and at least oneadditional MAb, preferably selected from the group consisting of mAbsreactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23,CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80,CD126, B7, MUC1, MUC2, MUC3, MUC4, Ia, HM1.24, and HLA-DR, tenascin,VEGF, PlGF, an oncogene, oncogene product, or a combination thereof,formulated in a pharmaceutically acceptable vehicle.

This therapeutic method can further be supplemented with theadministration to the subject concurrently or sequentially of atherapeutically effective amount of at least one therapeutic agent,formulated in a pharmaceutically acceptable vehicle, wherein thetherapeutic agent is preferably a cytotoxic agent, a radioactive label,an immunomodulator, a hormone, a photoactive therapeutic agent or acombination thereof, formulated in a pharmaceutically acceptablevehicle.

Further, the antibody fusion proteins can be administered to a subjectconcurrently or sequentially a therapeutically effective amount of atherapeutic conjugate comprising at least one MAb bound to at least onetherapeutic agent, wherein said MAb component of the conjugatepreferably comprises at least one humanized, chimeric, human or murine(for non-human subjects) MAb selected from the group consisting of a MAbreactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23,CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80,CD126, B7, MUC1, MUC2, MUC3, MUC4, Ia, HM1.24, and HLA-DR, tenascin,VEGF, PlGF, an oncogene, oncogene product, or a combination thereof,formulated in a pharmaceutically acceptable vehicle. The antibody fusionprotein itself can be conjugated to a therapeutic agent and thusprovides a vehicle to attach more than one therapeutic agent to anantibody component and these therapeutic agents can be a combination ofdifferent recited agents or combinations of the same agents, such as twodifferent therapeutic radioactive labels. Also encompassed by thepresent invention is a method of diagnosing a B-cell lymphoma orleukemia in a subject comprising administering to the subject, such as amammal, including humans and domestic and companion pets, such as dogs,cats, rabbits, guinea pigs, a diagnostic conjugate comprising ananti-CD20 MAb or fragment thereof or an antibody fusion protein orfragment thereof of the present invention that binds to the lymphoma orleukemia cell, wherein the anti-CD20 MAb or fragment thereof or antibodyfusion protein or fragment thereof is bound to at least one diagnosticagent, formulated in a pharmaceutically acceptable vehicle. The usefuldiagnostic agents are described herein.

2. Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of thepresent invention.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv and the like. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by theintact antibody. For example, an anti-CD20 monoclonal antibody fragmentbinds with an epitope of CD20. The term “antibody fragment” alsoincludes any synthetic or genetically engineered protein that acts likean antibody by binding to a specific antigen to form a complex. Forexample, antibody fragments include isolated fragments consisting of thevariable regions, such as the “Fv” fragments consisting of the variableregions of the heavy and light chains, recombinant single chainpolypeptide molecules in which light and heavy variable regions areconnected by a peptide linker (“scFv proteins”), and minimal recognitionunits consisting of the amino acid residues that mimic the hypervariableregion.

A naked antibody is generally an entire antibody which is not conjugatedto a therapeutic agent. This is so because the Fc portion of theantibody molecule provides effector functions, such as complementfixation and ADCC (antibody dependent cell cytotoxicity), which setmechanisms into action that may result in cell lysis. However, it ispossible that the Fc portion is not required for therapeutic function,with other mechanisms, such as apoptosis, coming into play. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric, humanized or humanantibodies.

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule is derived from those of ahuman antibody. For veterinary applications, the constant domains of thechimeric antibody may be derived from that of other species, such as acat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, is transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains. The constant domains of the antibodymolecule is derived from those of a human antibody.

A human antibody is an antibody obtained from transgenic mice that havebeen “engineered” to produce specific human antibodies in response toantigenic challenge. In this technique, elements of the human heavy andlight chain locus are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated intheir entirety by reference.

A therapeutic agent is a molecule or atom which is administeredseparately, concurrently or sequentially with an antibody moiety orconjugated to an antibody moiety, i.e., antibody or antibody fragment,or a subfragment, and is useful in the treatment of a disease. Examplesof therapeutic agents include antibodies, antibody fragments, drugs,toxins, nucleases, hormones, immunomodulators, chelators, boroncompounds, photoactive agents or dyes and radioisotopes.

A diagnostic agent is a molecule or atom which is administeredconjugated to an antibody moiety, i.e., antibody or antibody fragment,or subfragment, and is useful in diagnosing a disease by locating thecells containing the antigen. Useful diagnostic agents include, but arenot limited to, radioisotopes, dyes (such as with thebiotin-streptavidin complex), contrast agents, fluorescent compounds ormolecules and enhancing agents (e.g. paramagnetic ions) for magneticresonance imaging (MRI). U.S. Pat. No. 6,331,175 describes MRI techniqueand the preparation of antibodies conjugated to a MRI enhancing agentand is incorporated in its entirety by reference. Preferably, thediagnostic agents are selected from the group consisting ofradioisotopes, enhancing agents for use in magnetic resonance imaging,and fluorescent compounds. In order to load an antibody component withradioactive metals or paramagnetic ions, it may be necessary to react itwith a reagent having a long tail to which are attached a multiplicityof chelating groups for binding the ions. Such a tail can be a polymersuch as a polylysine, polysaccharide, or other derivatized orderivatizable chain having pendant groups to which can be boundchelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crownethers, bis-thiosemicarbazones, polyoximes, and like groups known to beuseful for this purpose. Chelates are coupled to the peptide antigensusing standard chemistries. The chelate is normally linked to theantibody by a group which enables formation of a bond to the moleculewith minimal loss of immunoreactivity and minimal aggregation and/orinternal cross-linking. other, more unusual, methods and reagents forconjugating chelates to antibodies are disclosed in U.S. Pat. No.4,824,659 to Hawthorne, entitled “Antibody Conjugates”, issued Apr. 25,1989, the disclosure of which is incorporated herein in its entirety byreference. Particularly useful metal-chelate combinations include2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used withdiagnostic isotopes in the general energy range of 60 to 4,000 keV, suchas ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁶²CU, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc,^(94m)Tc, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br, for radio-imaging. The same chelates,when complexed with non-radioactive metals, such as manganese, iron andgadolinium are useful for MRI, when used along with the antibodies ofthe invention. Macrocyclic chelates such as NOTA, DOTA, and TETA are ofuse with a variety of metals and radiometals, most particularly withradionuclides of gallium, yttrium and copper, respectively. Suchmetal-chelate complexes can be made very stable by tailoring the ringsize to the metal of interest. Other ring-type chelates such asmacrocyclic polyethers, which are of interest for stably bindingnuclides, such as ²²³Ra for RAIT are encompassed by the invention.

An immunoconjugate is a conjugate of an antibody component with atherapeutic or diagnostic agent. The diagnostic agent can comprise aradioactive or non-radioactive label, a contrast agent (such as formagnetic resonance imaging, computed tomography or ultrasound), and theradioactive label can be a gamma-, beta-, alpha-, Auger electron-, orpositron-emitting isotope.

An expression vector is a DNA molecules comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells, as well as an transgenicanimal, that have been genetically engineered to contain the clonedgene(s) in the chromosome or genome of the host cell or cells of thehost cells. Suitable mammalian host cells include myeloma cells, such asSP2/0 cells, and NS0 cells, as well as Chinese Hamster Ovary (CHO)cells, hybridoma cell lines and other mammalian host cell useful forexpressing antibodies. Also particularly useful to express mAbs andother fusion proteins, is a human cell line, PER.C6 disclosed in WO0063403 A2, which produces 2 to 200-fold more recombinant protein ascompared to conventional mammalian cell lines, such as CHO, COS, Vero,Hela, BHK and SP2-cell lines. Special transgenic animals with a modifiedimmune system are particularly useful for making fully human antibodies.

As used herein, the term antibody fusion protein is a recombinantlyproduced antigen-binding molecule in which two or more of the same ordifferent single-chain antibody or antibody fragment segments with thesame or different specificities are linked. Valency of the fusionprotein indicates how many binding arms or sites the fusion protein hasto a single antigen or epitope; i.e., monovalent, bivalent, trivalent ormutlivalent. The multivalency of the antibody fusion protein means thatit can take advantage of multiple interactions in binding to an antigen,thus increasing the avidity of binding to the antigen. Specificityindicates how many antigens or epitopes an antibody fusion protein isable to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Monospecific, multivalent fusionproteins have more than one binding site for an epitope but only bindswith one epitope, for example a diabody with two binding site reactivewith the same antigen. The fusion protein may comprise a single antibodycomponent, a multivalent or multispecific combination of differentantibody components or multiple copies of the same antibody component.The fusion protein may additionally comprise an antibody or an antibodyfragment and a therapeutic agent. Examples of therapeutic agentssuitable for such fusion proteins include immunomodulators(“antibody-immunomodulator fusion protein”) and toxins (“antibody-toxinfusion protein”). One preferred toxin comprises a ribonuclease (RNase),preferably a recombinant RNase.

A multispecific antibody is an antibody that can bind simultaneously toat least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. One specificity would be for aB-cell, T-cell, myeloid-, plasma-, and mast-cell antigen or epitope.Another specificity could be to a different antigen on the same celltype, such as CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, MUC1,and CD22 on B-cells. Multispecific, multivalent antibodies areconstructs that have more than one binding site, and the binding sitesare of different specificity. For example, a diabody, where one bindingsite reacts with one antigen and the other with the other antigen.

A bispecific antibody is an antibody that can bind simultaneously to twotargets which are of different structure. Bispecific antibodies (bsAb)and bispecific antibody fragments (bsFab) have at least one arm thatspecifically binds to, for example, a B-cell, T-cell, myeloid-, plasma-,and mast-cell antigen or epitope and at least one other arm thatspecifically binds to a targetable conjugate that bears a therapeutic ordiagnostic agent. A variety of bispecific fusion proteins can beproduced using molecular engineering. In one form, the bispecific fusionprotein is monovalent, consisting of, for example, a scFv with a singlebinding site for one antigen and a Fab fragment with a single bindingsite for a second antigen. In another form, the bispecific fusionprotein is divalent, consisting of, for example, an IgG with a bindingsite for one antigen and two scFv with two binding sites for a secondantigen.

Caninized or felinized antibodies are recombinant proteins in whichrodent (or another species) complementarity determining regions of amonoclonal antibody have been transferred from heavy and light variablechains of rodent (or another species) immunoglobulin into a dog or cat,respectively, immunoglobulin variable domain.

Domestic animals include large animals such as horses, cattle, sheep,goats, llamas, alpacas, and pigs, as well as companion animals. In apreferred embodiment, the domestic animal is a horse.

Companion animals include animals kept as pets. These are primarily dogsand cats, although small rodents, such as guinea pigs, hamsters, rats,and ferrets, are also included, as are subhuman primates such asmonkeys. In a preferred embodiment the companion animal is a dog or acat.

3. Preparation of Monoclonal Antibodies Including Chimeric, Humanizedand Human Antibodies

Monoclonal antibodies (MAbs) are a homogeneous population of antibodiesto a particular antigen and the antibody comprises only one type ofantigen binding site and binds to only one epitope on an antigenicdeterminant. Rodent monoclonal antibodies to specific antigens may beobtained by methods known to those skilled in the art. See, for example,Kohler and Milstein, Nature 256: 495 (1975), and Coligan et al. (eds.),CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley &Sons 1991) [hereinafter “Coligan”]. Briefly, monoclonal antibodies canbe obtained by injecting mice with a composition comprising an antigen,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B-lymphocytes, fusing theB-lymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones which produce antibodies to theantigen, culturing the clones that produce antibodies to the antigen,and isolating the antibodies from the hybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. For example,humanized monoclonal antibodies are produced by transferring mousecomplementary determining regions from heavy and light variable chainsof the mouse immunoglobulin into a human variable domain, and then,substituting human residues in the framework regions of the murinecounterparts. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions.

General techniques for cloning murine immunoglobulin variable domainsare described, for example, by the publication of Orlandi et al., Proc.Nat'l Acad. Sci. USA 86: 3833 (1989), which is incorporated by referencein its entirety. Techniques for constructing chimeric antibodies arewell known to those of skill in the art. As an example, Leung et al.,Hybridoma 13:469 (1994), describe how they produced an LL2 chimera bycombining DNA sequences encoding the V_(κ) and V_(H) domains of LL2monoclonal antibody, an anti-CD22 antibody, with respective human κ andIgG₁ constant region domains. This publication also provides thenucleotide sequences of the LL2 light and heavy chain variable regions,V_(κ) and V_(H), respectively. Techniques for producing humanized MAbsare described, for example, by Jones et al., Nature 321: 522 (1986),Riechmann et al., Nature 332: 323 (1988), Verhoeyen et al., Science 239:1534 (1988), Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992),Sandhu, Crit. Rev. Biotech. 12: 437 (1992), and Singer et al., J. Immun.150: 2844 (1993), each of which is hereby incorporated by reference.

A chimeric antibody is a recombinant protein that contains the variabledomains including the CDRs derived from one species of animal, such as arodent antibody, while the remainder of the antibody molecule; i.e., theconstant domains, is derived from a human antibody. Accordingly, achimeric monoclonal antibody can also be humanized by replacing thesequences of the murine FR in the variable domains of the chimeric MAbwith one or more different human FR. Specifically, mouse CDRs aretransferred from heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. As simply transferring mouse CDRs into human FRs often resultsin a reduction or even loss of antibody affinity, additionalmodification might be required in order to restore the original affinityof the murine antibody. This can be accomplished by the replacement ofone or more some human residues in the FR regions with their murinecounterparts to obtain an antibody that possesses good binding affinityto its epitope. See, for example, Tempest et al., Biotechnology 9:266(1991) and Verhoeyen et al., Science 239: 1534 (1988). Further, theaffinity of humanized, chimeric and human MAbs to a specific epitope canbe increased by mutagenesis of the CDRs, so that a lower dose ofantibody may be as effective as a higher dose of a lower affinity MAbprior to mutagenesis. See for example, WO0029584A1

Another method for producing the antibodies of the present invention isby production in the milk of transgenic livestock. See, e.g., Colman,A., Biochem. Soc. Symp., 63: 141-147, 1998; U.S. Pat. No. 5,827,690,both of which are incorporated in their entirety by reference. Two DNAconstructs are prepared which contain, respectively, DNA segmentsencoding paired immunoglobulin heavy and light chains. The DNA segmentsare cloned into expression vectors which contain a promoter sequencethat is preferentially expressed in mammary epithelial cells. Examplesinclude, but are not limited to, promoters from rabbit, cow and sheepcasein genes, the cow α-lactoglobulin gene, the sheep β-lactoglobulingene and the mouse whey acid protein gene. Preferably, the insertedfragment is flanked on its 3′ side by cognate genomic sequences from amammary-specific gene. This provides a polyadenylation site andtranscript-stabilizing sequences. The expression cassettes areco-injected into the pronuclei of fertilized, mammalian eggs, which arethen implanted into the uterus of a recipient female and allowed togestate. After birth, the progeny are screened for the presence of bothtransgenes by Southern analysis. In order for the antibody to bepresent, both heavy and light chain genes must be expressed concurrentlyin the same cell. Milk from transgenic females is analyzed for thepresence and functionality of the antibody or antibody fragment usingstandard immunological methods known in the art. The antibody can bepurified from the milk using standard methods known in the art.

A fully human antibody of the present invention, i.e., human anti-CD20MAbs or other human antibodies, such as anti-CD22, anti-CD19, anti-CD23,or anti-CD21 MAbs for combination therapy with humanized, chimeric orhuman anti-CD20 antibodies, can be obtained from a transgenic non-humananimal. See, e.g., Mendez et al., Nature Genetics, 15: 146-156 (1997);U.S. Pat. No. 5,633,425, which are incorporated in their entirety byreference. For example, a human antibody can be recovered from atransgenic mouse possessing human immunoglobulin loci. The mouse humoralimmune system is humanized by inactivating the endogenous immunoglobulingenes and introducing human immunoglobulin loci. The humanimmunoglobulin loci are exceedingly complex and comprise a large numberof discrete segments which together occupy almost 0.2% of the humangenome. To ensure that transgenic mice are capable of producing adequaterepertoires of antibodies, large portions of human heavy- andlight-chain loci must be introduced into the mouse genome. This isaccomplished in a stepwise process beginning with the formation of yeastartificial chromosomes (YACs) containing either human heavy- orlight-chain immunoglobulin loci in germline configuration. Since eachinsert is approximately 1 Mb in size, YAC construction requireshomologous recombination of overlapping fragments of the immunoglobulinloci. The two YACs, one containing the heavy-chain loci and onecontaining the light-chain loci, are introduced separately into mice viafusion of YAC-containing yeast spheroblasts with mouse embryonic stemcells. Embryonic stem cell clones are then microinjected into mouseblastocysts. Resulting chimeric males are screened for their ability totransmit the YAC through their germline and are bred with mice deficientin murine antibody production. Breeding the two transgenic strains, onecontaining the human heavy-chain loci and the other containing the humanlight-chain loci, creates progeny which produce human antibodies inresponse to immunization.

Further recent methods for producing bispecific mAbs include engineeredrecombinant mAbs which have additional cysteine residues so that theycrosslink more strongly than the more common immunoglobulin isotypes.See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225, 1997.Another approach is to engineer recombinant fusion proteins linking twoor more different single-chain antibody or antibody fragment segmentswith the needed dual specificities. See, e.g., Coloma et al., NatureBiotech. 15:159-163, 1997. A variety of bispecific fusion proteins canbe produced using molecular engineering. In one form, the bispecificfusion protein is monovalent, consisting of, for example, a scFv with asingle binding site for one antigen and a Fab fragment with a singlebinding site for a second antigen. In another form, the bispecificfusion protein is divalent, consisting of, for example, an IgG with twobinding sites for one antigen and two scFv with two binding sites for asecond antigen.

Bispecific fusion proteins linking two or more different single-chainantibodies or antibody fragments are produced in similar manner.Recombinant methods can be used to produce a variety of fusion proteins.For example a fusion protein comprising a Fab fragment derived from ahumanized monoclonal anti-CD20 antibody and a scFv derived from a murineanti-diDTPA can be produced. A flexible linker, such as GGGS (SEQ ID NO:55) connects the scFv to the constant region of the heavy chain of theanti-CD20 antibody. Alternatively, the scFv can be connected to theconstant region of the light chain of another humanized antibody.Appropriate linker sequences necessary for the in-frame connection ofthe heavy chain Fd to the scFv are introduced into the VL and VK domainsthrough PCR reactions. The DNA fragment encoding the scFv is thenligated into a staging vector containing a DNA sequence encoding the CH1domain. The resulting scFv-CH₁ construct is excised and ligated into avector containing a DNA sequence encoding the VH region of an anti-CD20antibody. The resulting vector can be used to transfect an appropriatehost cell, such as a mammalian cell for the expression of the bispecificfusion protein.

4. Production of Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. The antibody fragments are antigen binding portions ofan antibody, such as F(ab)₂, Fab′, Fab, Fv, sFv and the like. Otherantibody fragments include, but are not limited to: the F(ab)′₂fragments which can be produced by pepsin digestion of the antibodymolecule and the Fab′ fragments, which can be generated by reducingdisulfide bridges of the F(ab)′₂ fragments. Alternatively, Fab′expression libraries can be constructed (Huse et al., 1989, Science,246:1274-1281) to allow rapid and easy identification of monoclonal Fab′fragments with the desired specificity. The present inventionencompasses antibodies and antibody fragments.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). A scFvmolecule is denoted as either VL-L-VH if the VL domain is the N-terminalpart of the scFv molecule, or as VH-L-VL if the VH domain is theN-terminal part of the scFv molecule. Methods for making scFv moleculesand designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow, “SingleChain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker,“Single Chain Antibody Variable Regions,” TIBTECH, Vol 9: 132-137(1991). These references are incorporated herein by reference.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment denotedF(ab′)₂. This fragment can be further cleaved using a thiol reducingagent, and optionally a blocking group for the sulfhydryl groupsresulting from cleavage of disulfide linkages, to produce 3.5S Fab′monovalent fragments. Alternatively, an enzymatic cleavage using papainproduces two monovalent Fab fragments and an Fc fragment directly. Thesemethods are described, for example, by Goldenberg, U.S. Pat. Nos.4,036,945 and 4,331,647 and references contained therein, which patentsare incorporated herein in their entireties by reference. Also, seeNisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem.J. 73: 119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). A CDR is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to ashypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody-producing cells. See, for example, Larrick et al., Methods: ACompanion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,” in MONOCLONALANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

5. Multispecific and Multivalent Antibodies

The anti-CD20 antibodies, as well as other antibodies with differentspecificities for use in combination therapy, described herein, can alsobe made as multispecific antibodies (comprising at least one bindingsite to a CD20 epitope or antigen and at least one binding site toanother epitope on CD20 or another antigen) and multivalent antibodies(comprising multiple binding sites to the same epitope or antigen).Multivalent target binding proteins are described in U.S. Ser. No.09/911,610 (Leung et al.), which is incorporated herein by reference inits entirety.

The present invention provides a bispecific antibody or antibodyfragment having at least a binding region that specifically binds atargeted cell marker and at least one other binding region thatspecifically binds a targetable conjugate. The targetable conjugatecomprises a carrier portion which comprises or bears at least oneepitope recognized by at least one binding region of the bispecificantibody or antibody fragment.

A variety of recombinant methods can be used to produce bispecificantibodies and antibody fragments as described above.

An anti-CD20 multivalent antibody is also contemplated in the presentinvention. This multivalent target binding protein is constructed byassociation of a first and a second polypeptide. The first polypeptidecomprises a first single chain Fv molecule covalently linked to a firstimmunoglobulin-like domain which preferably is an immunoglobulin lightchain variable region domain. The second polypeptide comprises a secondsingle chain Fv molecule covalently linked to a secondimmunoglobulin-like domain which preferably is an immunoglobulin heavychain variable region domain. Each of the first and second single chainFv molecules forms a target binding site, and the first and secondimmunoglobulin-like domains associate to form a third target bindingsite.

A single chain Fv molecule with the VL-L-VH configuration, wherein L isa linker, may associate with another single chain Fv molecule with theVH-L-VL configuration to form a bivalent dimer. In this case, the VLdomain of the first scFv and the VH domain of the second scFv moleculeassociate to form one target binding site, while the VH domain of thefirst scFv and the VL domain of the second scFv associate to form theother target binding site.

Another embodiment of the present invention is a CD20 bispecific,trivalent targeting protein comprising two heterologous polypeptidechains associated non-covalently to form three binding sites, two ofwhich have affinity for one target and a third which has affinity for ahapten that can be made and attached to a carrier for a diagnosticand/or therapeutic agent. Preferably, the binding protein has two CD20binding sites and one CD22 binding site. The bispecific, trivalenttargeting agents have two different scFvs, one scFv contains two V_(H)domains from one antibody connected by a short linker to the V_(L)domain of another antibody and the second scFv contains two V_(L)domains from the first antibody connected by a short linker to the V_(H)domain of the other antibody. The methods for generating multivalent,multispecific agents from V_(H) and V_(L) domains provide thatindividual chains synthesized from a DNA plasmid in a host organism arecomposed entirely of V_(H) domains (the V_(H)-chain) or entirely ofV_(L) domains (the V_(L)-chain) in such a way that any agent ofmultivalency and multispecificity can be produced by non-covalentassociation of one V_(H)-chain with one V_(L)-chain. For example,forming a trivalent, trispecific agent, the V_(H)-chain will consist ofthe amino acid sequences of three V_(H) domains, each from an antibodyof different specificity, joined by peptide linkers of variable lengths,and the V_(L)-chain will consist of complementary V_(L) domains, joinedby peptide linkers similar to those used for the V_(H)-chain. Since theV_(H) and V_(L) domains of antibodies associate in an anti-parallelfashion, the preferred method in this invention has the V_(L) domains inthe V_(L)-chain arranged in the reverse order of the V_(H) domains inthe V_(H)-chain.

6. Diabodies, Triabodies and Tetrabodies

The anti-CD20 antibodies of the present invention can also be used toprepare functional bispecific single-chain antibodies (bscAb), alsocalled diabodies, and can be produced in mammalian cells usingrecombinant methods. See, e.g., Mack et al., Proc. Natl. Acad. Sci., 92:7021-7025, 1995, incorporated. For example, bscAb are produced byjoining two single-chain Fv fragments via a glycine-serine linker usingrecombinant methods. The V light-chain (V_(L)) and V heavy-chain (V_(H))domains of two antibodies of interest are isolated using standard PCRmethods. The V_(L) and V_(H) cDNA's obtained from each hybridoma arethen joined to form a single-chain fragment in a two-step fusion PCR.The first PCR step introduces the (Gly₄-Ser₁)₃ linker, and the secondstep joins the V_(L) and V_(H) amplicons. Each single chain molecule isthen cloned into a bacterial expression vector. Following amplification,one of the single-chain molecules is excised and sub-cloned into theother vector, containing the second single-chain molecule of interest.The resulting bscAb fragment is subcloned into an eukaryotic expressionvector. Functional protein expression can be obtained by transfectingthe vector into chinese hamster ovary cells. Bispecific fusion proteinsare prepared in a similar manner. Bispecific single-chain antibodies andbispecific fusion proteins are included within the scope of the presentinvention.

For example, a humanized, chimeric or human anti-CD20 monoclonalantibody can be used to produce antigen specific diabodies, triabodies,and tetrabodies. The monospecific diabodies, triabodies, and tetrabodiesbind selectively to targeted antigens and as the number of binding siteson the molecule increases, the affinity for the target cell increasesand a longer residence time is observed at the desired location. Fordiabodies, the two chains comprising the V_(H) polypeptide of thehumanized CD20 MAb connected to the V_(K) polypeptide of the humanizedCD20 MAb by a five amino acid residue linker are utilized. Each chainforms one half of the humanized CD20 diabody. In the case of triabodies,the three chains comprising V_(H) polypeptide of the humanized CD20 MAbconnected to the V_(K) polypeptide of the humanized CD20 MAb by nolinker are utilized. Each chain forms one third of the hCD20 triabody.

The ultimate use of the bispecific diabodies described herein is forpre-targeting CD20 positive tumors for subsequent specific delivery ofdiagnostic or therapeutic agents. These diabodies bind selectively totargeted antigens allowing for increased affinity and a longer residencetime at the desired location. Moreover, non-antigen bound diabodies arecleared from the body quickly and exposure of normal tissues isminimized Bispecific antibody point mutations for enhancing the rate ofclearance can be found in U.S. Provisional Application No. 60/361,037 toQu et al. (Atty Docket No. 18733/1037), which is incorporated herein byreference in its entirety. Bispecific diabodies for affinity enhancementare disclosed in U.S. application Ser. Nos. 10/270,071 (Rossi et al.),10/270,073 (Rossi et al.) and 10/328,190 (Rossi et al.), which areincorporated herein by reference in their entirety. The diagnostic andtherapeutic agents can include isotopes, drugs, toxins, cytokines,hormones, growth factors, conjugates, radionuclides, and metals. Forexample, gadolinium metal is used for magnetic resonance imaging (MRI).Examples of radionuclides are ²²⁵Ac, ¹⁸F, ⁶⁸Ga, ⁶⁷Ga, ⁹⁰Y, ⁸⁶Y, ¹¹¹In,¹²⁵I, ¹²³I, ^(99m) Tc, ^(94m)Tc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,²¹²Bi, ²¹³Bi, ³²P, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br, and ²¹¹At. Other radionuclidesare also available as diagnostic and therapeutic agents, especiallythose in the energy range of 60 to 4,000 keV.

More recently, a tetravalent tandem diabody (termed tandab) with dualspecificity has also been reported (Cochlovius et al., Cancer Research(2000) 60: 4336-4341). The bispecific tandab is a dimer of two identicalpolypeptides, each containing four variable domains of two differentantibodies (V_(H1), V_(L1), V_(H2), V_(L2)) linked in an orientation tofacilitate the formation of two potential binding sites for each of thetwo different specificities upon self-association.

7. Conjugated Multivalent and Multispecific Anti-CD20 Antibodies

In another embodiment of the instant invention is a conjugatedmultivalent anti-CD20 antibody. Additional amino acid residues may beadded to either the N- or C-terminus of the first or the secondpolypeptide. The additional amino acid residues may comprise a peptidetag, a signal peptide, a cytokine, an enzyme (for example, a pro-drugactivating enzyme), a hormone, a peptide toxin, such as pseudomonasextoxin, a peptide drug, a cytotoxic protein or other functionalproteins. As used herein, a functional protein is a protein which has abiological function.

In one embodiment, drugs, toxins, radioactive compounds, enzymes,hormones, cytotoxic proteins, chelates, cytokines and other functionalagents may be conjugated to the multivalent target binding protein,preferably through covalent attachments to the side chains of the aminoacid residues of the multivalent target binding protein, for exampleamine, carboxyl, phenyl, thiol or hydroxyl groups. Various conventionallinkers may be used for this purpose, for example, diisocyanates,diisothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides,maleimide-hydroxysuccinimide esters, glutaraldehyde and the like.Conjugation of agents to the multivalent protein preferably does notsignificantly affect the protein's binding specificity or affinity toits target. As used herein, a functional agent is an agent which has abiological function. A preferred functional agent is a cytotoxic agent.

In still other embodiments, bispecific antibody-directed delivery oftherapeutics or prodrug polymers to in vivo targets can be combined withbispecific antibody delivery of radionuclides, such that combinationchemotherapy and radioimmunotherapy is achieved. Each therapy can beconjugated to the targetable conjugate and administered simultaneously,or the nuclide can be given as part of a first targetable conjugate andthe drug given in a later step as part of a second targetable conjugate.

In another embodiment, cytotoxic agents may be conjugated to a polymericcarrier, and the polymeric carrier may subsequently be conjugated to themultivalent target binding protein. For this method, see Ryser et al.,Proc. Natl. Acad. Sci. USA, 75:3867-3870, 1978, U.S. Pat. No. 4,699,784and U.S. Pat. No. 4,046,722, which are incorporated herein by reference.Conjugation preferably does not significantly affect the bindingspecificity or affinity of the multivalent binding protein.

8. Humanized, Chimeric and Human Antibodies Use for Treatment andDiagnosis

Humanized, chimeric and human monoclonal antibodies, i.e., anti-CD20MAbs and other MAbs described herein, in accordance with this inventionare suitable for use in therapeutic methods and diagnostic methods.Accordingly, the present invention contemplates the administration ofthe humanized, chimeric and human antibodies of the present inventionalone as a naked antibody or administered as a multimodal therapy,temporally according to a dosing regimen, but not conjugated to, atherapeutic agent. The efficacy of the naked anti-CD20 MAbs can beenhanced by supplementing naked antibodies with one or more other nakedantibodies, i.e., MAbs to specific antigens, such as CD4, CD5, CD8,CD14, CD15, CD19, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L,CD46, CD52, CD54, CD74, CD80, CD126, B7, MUC1, Ia, HM1.24, or HLA-DR,tenascin, VEGF, PlGF, an oncogene, an oncogene product, or a combinationthereofwith one or more immunoconjugates of anti-CD20, or antibodies totheses recited antigens, conjugated with therapeutic agents, includingdrugs, toxins, immunomodulators, hormones, therapeutic radionuclides,etc., with one or more therapeutic agents, including drugs,oligonucleotide, toxins, immunomodulators, hormones, therapeuticradionuclides, etc., administered concurrently or sequentially oraccording to a prescribed dosing regimen, with the MAbs. PreferredB-cell antigens include those equivalent to human CD19, CD20, CD21,CD22, CD23, CD46, CD52, CD74, CD80, and CD5 antigens. Preferred T-cellantigens include those equivalent to human CD4, CD8 and CD25 (the IL-2receptor) antigens. An equivalent to HLA-DR antigen can be used intreatment of both B-cell and T-cell disorders. Particularly preferredB-cell antigens are those equivalent to human CD19, CD22, CD21, CD23,CD74, CD80, and HLA-DR antigens. Particularly preferred T-cell antigensare those equivalent to human CD4, CD8 and CD25 antigens. CD46 is anantigen on the surface of cancer cells that block complement-dependentlysis (CDC).

Further, the present invention contemplates the administration of animmunoconjugate for diagnostic and therapeutic uses in B cell lymphomasand other disease or disorders. An immunoconjugate, as described herein,is a molecule comprising an antibody component and a therapeutic ordiagnostic agent, including a peptide which may bear the diagnostic ortherapeutic agent. An immunoconjugate retains the immunoreactivity ofthe antibody component, i.e., the antibody moiety has about the same orslightly reduced ability to bind the cognate antigen after conjugationas before conjugation.

A wide variety of diagnostic and therapeutic agents can beadvantageously conjugated to the antibodies of the invention. Thetherapeutic agents recited here are those agents that also are usefulfor administration separately with the naked antibody as describedabove. Therapeutic agents include, for example, chemotherapeutic drugssuch as vinca alkaloids, anthracyclines, epidophyllotoxin, taxanes,antimetabolites, alkylating agents, antikinase agents, antibiotics,Cox-2 inhibitors, antimitotics, antiangiogenic and apoptotoic agents,particularly doxorubicin, methotrexate, taxol, CPT-11, camptothecans,and others from these and other classes of anticancer agents, and thelike. Other useful cancer chemotherapeutic drugs for the preparation ofimmunoconjugates and antibody fusion proteins include nitrogen mustards,alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2inhibitors, pyrimidine analogs, purine analogs, platinum coordinationcomplexes, hormones, and the like. Suitable chemotherapeutic agents aredescribed in REMINGTON′S PHARMACEUTICAL SCIENCES, 19th Ed. (MackPublishing Co. 1995), and in GOODMAN AND GILMAN′S THE PHARMACOLOGICALBASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as wellas revised editions of these publications. Other suitablechemotherapeutic agents, such as experimental drugs, are known to thoseof skill in the art.

Additionally, a chelator such as DTPA (such as Mx-DTPA), DOTA, TETA, orNOTA or a suitable peptide, to which a detectable label, such as afluorescent molecule, or cytotoxic agent, such as a heavy metal orradionuclide, can be conjugated. For example, a diagnostically ortherapeutically useful immunoconjugate can be obtained by conjugating aphotoactive agent or dye to an antibody composite. Fluorescentcompositions, such as fluorochrome, and other chromogens, or dyes, suchas porphyrins sensitive to visible light, have been used to detect andto treat lesions by directing the suitable light to the lesion. Intherapy, this has been termed photoradiation, phototherapy, orphotodynamic therapy (Jori et al. (eds.), PHOTODYNAMIC THERAPY OF TUMORSAND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.Britain 22:430 (1986)). Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy. Mew et al.,J. Immunol. 130:1473 (1983); idem., Cancer Res. 45:4380 (1985); Oseroffet al., Proc. Natl. Acad. Sci. USA 83:8744 (1986); idem., Photochem.Photobiol. 46:83 (1987); Hasan et al., Prog. Clin. Biol. Res. 288:471(1989); Tatsuta et al., Lasers Surg. Med. 9:422 (1989); Pelegrin et al.,Cancer 67:2529 (1991). However, these earlier studies did not includeuse of endoscopic therapy applications, especially with the use ofantibody fragments or subfragments. Thus, the present inventioncontemplates the therapeutic use of immunoconjugates comprisingphotoactive agents or dyes.

Also contemplated by the present invention are the use of radioactiveand non-radioactive agents as diagnostic agents. A suitablenon-radioactive diagnostic agent is a contrast agent suitable formagnetic resonance imaging, computed tomography or ultrasound. Magneticimaging agents include, for example, non-radioactive metals, such asmanganese, iron and gadolinium, complexed with metal-chelatecombinations that include 2-benzyl-DTPA and its monomethyl andcyclohexyl analogs, when used along with the antibodies of theinvention. See U.S. Ser. No. 09/921,290 filed on Oct. 10, 2001, which isincorporated in its entirety by reference.

Furthermore, a radiolabeled antibody or immunoconjugate may comprise a[gamma]-emitting radioisotope or a positron-emitter useful fordiagnostic imaging. Suitable radioisotopes, particularly in the energyrange of 60 to 4,000 keV, include ¹³¹I, ¹²¹I, ¹²⁴I, ⁸⁶Y, ⁶²Cu, ⁶⁴Cu,¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ¹⁸F, ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br, and thelike. See for example, U.S. patent application entitled “LabelingTargeting Agents with Gallium-68”-Inventors G. L. Griffiths and W. J.McBride, (U.S. Ser. No. 10/318,401, which claims benefit of U.S.Provisional Application No. 60/342,104, and now has issued as U.S. Pat.No. 7,011,816), which discloses positron emitters, such as ¹⁸F, ⁶⁸Ga,^(94m)Tc and the like, for imaging purposes and which is incorporated inits entirety by reference. Particularly useful therapeutic radionuclidesinclude, but are not limited to, ³²P, ³³P, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁹⁰Y,¹¹¹Ag, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁷Lu,¹⁸⁶Re, ¹⁸⁸Re. ¹⁸⁹Re, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra and ²²⁵Ac. Particularlyuseful diagnostic/detection radionuclides include, but are not limitedto, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc,^(99m)Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P, ⁹⁰Y, ¹⁸⁸Re, and¹⁷⁵Lu.

A toxin, such as Pseudomonas exotoxin, may also be complexed to or formthe therapeutic agent portion of an antibody fusion protein of ananti-CD20 antibody of the present invention. Other toxins suitablyemployed in the preparation of such conjugates or other fusion proteins,include ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example,Pastan et al., Cell 47:641 (1986), and Goldenberg, C A—A Cancer Journalfor Clinicians 44:43 (1994). Additional toxins suitable for use in thepresent invention are known to those of skill in the art and aredisclosed in U.S. Pat. No. 6,077,499, which is incorporated in itsentirety by reference.

An immunomodulator, such as a cytokine may also be conjugated to, orform the therapeutic agent portion of an antibody fusion protein or beadministered with the humanized anti-CD20 antibodies of the presentinvention. Suitable cytokines for the present invention include, but arenot limited to, interferons and interleukins, as described below.

An oligonucleotide, such the antisense molecules inhibiting bcl-2expression that are described in U.S. Pat. No. 5,734,033 (Reed) which isincorporated by reference in its entirety, may also be conjugated to, orform the therapeutic agent portion of an antibody fusion protein or beadministered with the humanized anti-CD20 antibodies of the presentinvention.

9. Preparation of Immunoconjugates

Any of the antibodies or antibody fusion proteins of the presentinvention can be conjugated with one or more therapeutic or diagnosticagents. Generally, one therapeutic or diagnostic agent is attached toeach antibody or antibody fragment but more than one therapeutic agentor diagnostic agent can be attached to the same antibody or antibodyfragment. The antibody fusion proteins of the present invention comprisetwo or more antibodies or fragments thereof and each of the antibodiesthat composes this fusion protein can contain a therapeutic agent ordiagnostic agent. Additionally, one or more of the antibodies of theantibody fusion protein can have more than one therapeutic of diagnosticagent attached. Further, the therapeutic agents do not need to be thesame but can be different therapeutic agents. For example, one canattach a drug and a radioisotope to the same fusion protein.Particularly, an IgG can be radiolabeled with ¹³¹I and attached to adrug. The ¹³¹I can be incorporated into the tyrosine of the IgG and thedrug attached to the epsilon amino group of the IgG lysines. Boththerapeutic and diagnostic agents also can be attached to reduced SHgroups and to the carbohydrate side chains.

Radionuclides suitable for treating a disease tissue substantially decayby beta-particle emission and include, but are not limited to: ³²P, ³³P,⁴⁷Sc, ⁵⁹Fe, ⁶⁴Cu, ⁶⁷Cu, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁰Y, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd,¹¹¹Ag, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁹Er,¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹Pb, ²¹²Pb and ²¹³Bi.Maximum decay energies of useful beta-particle-emitting nuclides arepreferably 20-5,000 keV, more preferably 100-4,000 keV, and mostpreferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, ⁵⁸Co,⁶⁷Ga, ^(80m)Br, ^(99m)Tc, ^(103m)Rh, ¹⁰⁹Pt, ¹¹¹In, ¹¹⁹Sb, ¹²⁵I, ¹⁶¹Ho,^(189m)Os and ¹⁹²Ir. Decay energies of useful Auger-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: ¹⁵²Dy, ²¹¹At, ²¹²Bi, ²²³Ra, ²¹⁹Rn, ²¹⁵Po, ²¹¹Bi,²²⁵Ac, ²²¹Fr, ²¹⁷At, ²¹³Bi and ²⁵⁵Fm. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.

Radionuclides useful as diagnostic agents utilizing gamma-ray detectioninclude, but are not limited to: ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu, ⁶⁷Ga,⁷⁵Se, ⁹⁷Ru, ^(99m)Tc, ¹¹¹In, ^(114m)In, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁶⁹Yb, ¹⁹⁷Hg,and ²⁰¹Tl. Decay energies of useful gamma-ray emitting radionuclides arepreferably 20-2000 keV, more preferably 60-600 keV, and most preferably100-300 keV.

Radionuclides useful for positron emission tomography include, but arenot limited to: ¹⁸F, ⁵¹Mn, ^(52m)Mn, ⁵²Fe, ⁵⁵Co, ⁶²Cu, ⁶⁴Cu, ⁶⁸Ga, ⁷²As,⁷⁵Br, ⁷⁶Br, ^(82m)Rb, ⁸³Sr, ⁸⁶Y, ⁸⁹Zr, 94mTc, ¹¹⁰In, ¹²⁰I and ¹²⁴I.Total decay energies of useful positron-emitting radionuclides arepreferably <2,000 keV, more preferably under 1,000 keV, and mostpreferably <700 keV.

Bispecific antibodies of the present invention are useful inpretargeting methods and provide a preferred way to deliver twotherapeutic agents or two diagnostic agents to a subject. U.S. Ser. Nos.09/382,186 and 09/337,756, now U.S. Pat. Nos. 7,052,872 and 7,074,405,respectively, disclose a method of pretargeting using a bispecificantibody, in which the bispecific antibody is labeled with ¹²⁵I anddelivered to a subject, followed by a divalent peptide labeled with^(99m)Tc, and are incorporated herein by reference in their entirety.Pretargeting methods are also described in U.S. Ser. Nos. 09/823,746(now U.S. Pat. No. 6,962,702—Hansen et al.) and 10/150,654 (now U.S.Pat. No. 7,138,103—Goldenberg et al.), and U.S. Provisional Application60/444,357, filed Jan. 31, 2003, entitled “Methods and Compositions forAdministration of Therapeutic and Diagnostic Agents, Atty Docket No.018733/1103 (McBride et al.), which are all also incorporated herein byreference in their entirety. The delivery results in excellenttumor/normal tissue ratios for ¹²⁵I and ^(99m)Tc, thus showing theutility of two diagnostic radioisotopes. Any combination of knowntherapeutic agents or diagnostic agents can be used to label theantibodies and antibody fusion proteins. The binding specificity of theantibody component of the MAb conjugate, the efficacy of the therapeuticagent or diagnostic agent and the effector activity of the Fc portion ofthe antibody can be determined by standard testing of the conjugates.

The invention is directed to a method for pretargeting a cell in apatients suffering from a B-cell lymphoma or leukemia or an autoimmunedisease comprising:

(i) administering an antibody fusion protein or fragment thereof that ismultispecific having at least one arm that specifically binds the celland at least one other arm that specifically binds a targetableconjugate; (ii) optionally, administering to the patient a clearingcomposition, and allowing the composition to clear non-antigen boundantibody fusion protein or fragment thereof from circulation; and (iii)administering to the patient a targetable conjugate comprising a carrierportion which comprises or bears at least one epitope recognizable by atleast one other arm of the antibody fusion protein or fragment thereof,and is conjugated at least one first therapeutic or diagnostic agent.The antibody fusion protein of the present invention should bemultispecific antibody. In a preferred embodiment the antibody is abispecific antibody, and can be a diabody. The first therapeutic agentis selected from the group consisting of a radioactive label, animmunomodulator, a hormone, a photoactive therapeutic agent, a cytotoxicagent, an oligonucleotide and a combination thereof and wherein thefirst diagnostic agent is at least one of a radioactive label, aphotoactive diagnostic agent or a non-radioactive label.

The antibody fusion protein or fragment thereof also may be conjugatedto a second therapeutic, such as at least one radioactive label, animmunomodulator, a hormone, a photoactive therapeutic agent, a cytotoxicagent, an oligonucleotide and a combination thereof or may be conjugatedthe second diagnostic agent, such as at least one of a radioactivelabel, a photoactive diagnostic agent or a non-radioactive label. In oneembodiment, the first and second therapeutic agent or diagnostic agentare the same.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation. As analternative, such peptides can be attached to the antibody componentusing a heterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the therapeutic ordiagnostic agent can be conjugated via a carbohydrate moiety in the Fcregion of the antibody. The carbohydrate group can be used to increasethe loading of the same peptide that is bound to a thiol group, or thecarbohydrate moiety can be used to bind a different peptide.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, all of which are incorporated in their entirety by reference.The general method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality of peptide.This reaction results in an initial Schiff base (imine) linkage, whichcan be stabilized by reduction to a secondary amine to form the finalconjugate.

The Fc region is absent if the antibody used as the antibody componentof the immunoconjugate is an antibody fragment. However, it is possibleto introduce a carbohydrate moiety into the light chain variable regionof a full length antibody or antibody fragment. See, for example, Leunget al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S. Pat. No.5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868, all of whichare incorporated in their entirety by reference. The engineeredcarbohydrate moiety is used to attach the therapeutic or diagnosticagent.

10. Pharmaceutically Acceptable Excipients

The humanized, chimeric and human anti-CD20 mAbs to be delivered to asubject can consist of the MAb alone, immunoconjugate, fusion protein,or can comprise one or more pharmaceutically suitable excipients, one ormore additional ingredients, or some combination of these.

The immunoconjugate or naked antibody of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the immunoconjugate or naked antibody are combinedin a mixture with a pharmaceutically suitable excipient. Sterilephosphate-buffered saline is one example of a pharmaceutically suitableexcipient. Other suitable excipients are well-known to those in the art.See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUGDELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON′S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

The immunoconjugate or naked antibody of the present invention can beformulated for intravenous administration via, for example, bolusinjection or continuous infusion. Preferably, the antibody of thepresent invention is infused over a period of less than about 4 hours,and more preferably, over a period of less than about 3 hours. Forexample, the first 25-50 mg could be infused within 30 minutes,preferably even 15 min, and the remainder infused over the next 2-3 hrs.Formulations for injection can be presented in unit dosage form, e.g.,in ampules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic or diagnostic conjugate or nakedantibody. Control release preparations can be prepared through the useof polymers to complex or adsorb the immunoconjugate or naked antibody.For example, biocompatible polymers include matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. Sherwood et al.,Bio/Technology 10: 1446 (1992). The rate of release of animmunoconjugate or antibody from such a matrix depends upon themolecular weight of the immunoconjugate or antibody, the amount ofimmunoconjugate, antibody within the matrix, and the size of dispersedparticles. Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al.,supra. Other solid dosage forms are described in Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The immunoconjugate, antibody fusion proteins, or naked antibody mayalso be administered to a mammal subcutaneously or even by otherparenteral routes. Moreover, the administration may be by continuousinfusion or by single or multiple boluses. Preferably, the antibody ofthe present invention us infused over a period of less than about 4hours, and more preferably, over a period of less than about 3 hours.This is preferably performed by infusing slowly at first. For example, adose of 25 to 50 mg is infused within 15-30 minutes and the remainder ofthe dose is infused over a period of up to 2-3 hrs. In general, thedosage of an administered immunoconjugate, fusion protein or nakedantibody for humans will vary depending upon such factors as thepatient's age, weight, height, sex, general medical condition andprevious medical history. Typically, it is desirable to provide therecipient with a dosage of immunoconjugate, antibody fusion protein ornaked antibody that is in the range of from about 1 mg/kg to 20 mg/kg asa single intravenous infusion, although a lower or higher dosage alsomay be administered as circumstances dictate. Therefore, 1-20 mg/kg fora 70 kg patient, for example, is a dose of 70-1,400 mg, or 41-824 mg/m²for a 1.7-m patient. This dosage may be repeated as needed, for example,once per week for 4-10 weeks, preferably once per week for 8 weeks, andmore preferably, once per week for 4 weeks. It may also be given lessfrequently, such as every other week for several months. Morespecifically, an antibody of the present invention, such as nakedanti-CD20, may be administered as one dosage every 2 or 3 weeks,repeated for a total of at least 3 dosages. Also preferred, theantibodies of the present invention may be administered once per weekfor 4-8 weeks. In other words, if the dosage is lowered to approximately200-300 mg/m² (which is 340 mg per dosage for a 1.7-m patient, or 4.9mg/kg for a 70 kg patient), it may be administered once weekly for 4 to8 weeks. Alternatively, the dosage schedule may be decreased, namelyevery 2 or 3 weeks for 2-3 months; for example, if the dosage is 300-500mg/m² (i.e., 510-850 mg for a 1.7-m patient, or 7.3-12 mg/kg for a 70 kgpatient). The dosing schedule can optionally be repeated at otherintervals and dosage may be given through various parenteral routes,with appropriate adjustment of the dose and schedule.

For purposes of therapy, the immunoconjugate, fusion protein, or nakedantibody is administered to a mammal in a therapeutically effectiveamount. A suitable subject for the present invention are usually ahuman, although a non-human animal subject is also contemplated. Anantibody preparation is said to be administered in a “therapeuticallyeffective amount” if the amount administered is physiologicallysignificant. An agent is physiologically significant if its presenceresults in a detectable change in the physiology of a recipient mammal.In particular, an antibody preparation of the present invention isphysiologically significant if its presence invokes an antitumorresponse or mitigates the signs and symptoms of an autoimmune diseasestate. A physiologically significant effect could also be the evocationof a humoral and/or cellular immune response in the recipient mammal.

11. Methods of Treatment

The present invention contemplates the use of naked anti-CD20 antibodiesof the present invention as the primary composition for treatment of Bcell disorders and other diseases. In particular, the compositionsdescribed herein are particularly useful for treatment of variousautoimmune as well as indolent forms of B-cell lymphomas, aggressiveforms of B-cell lymphomas, chronic lymphatic leukemias, acute lymphaticleukemias, and Waldenström's macroglobulinemia. For example, thehumanized anti-CD20 antibody components and immunoconjugates can be usedto treat both indolent and aggressive forms of non-Hodgkin's lymphoma.

The compositions for treatment contain at least one humanized, chimericor human monoclonal anti-CD20 antibody alone or in combination withother antibodies, such as other humanized, chimeric, or humanantibodies, therapeutic agents or immunomodulators. In particular,combination therapy with a fully human antibody is also contemplated andis produced by the methods as set forth above.

Naked or conjugated antibodies to the same or different epitope orantigen may be also be combined with one or more of the antibodies ofthe present invention. For example, a humanized, chimeric or human nakedanti-CD20 antibody may be combined with another naked humanized, nakedchimeric or naked human anti-CD20, a humanized, chimeric or human nakedanti-CD20 antibody may be combined with an anti-CD20 immunoconjugate, anaked anti-CD20 antibody may be combined with an anti-CD22radioconjugate or an anti-CD22 naked antibody may be combined with ahumanized, chimeric or human anti-CD20 antibody conjugated to anisotope, or one or more chemotherapeutic agents, cytokines, toxins or acombination thereof. A fusion protein of a humanized, chimeric or humanCD20 antibody and a toxin or immunomodulator, or a fusion protein of atleast two different B-cell antibodies (e.g., a CD20 and a CD22 MAb) mayalso be used in this invention. Many different antibody combinations,targeting at least two different antigens associated with B-celldisorders, as listed already above, may be constructed, either as nakedantibodies or as partly naked and partly conjugated with a therapeuticagent or immunomodulator, or merely in combination with anothertherapeutic agents, such as a cytotoxic drug or with radiation.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, such as tumor necrosis factor (TNF), andhematopoietic factors, such as interleukins (e.g., interleukin-1 (IL-1),IL-2, IL-3, IL-6, IL-10, IL-12, IL-21 and IL-18), colony stimulatingfactors (e.g., granulocyte-colony stimulating factor (G-CSF) andgranulocyte macrophage-colony stimulating factor (GM-CSF)), interferons(e.g., interferons-α, -β and -γ), the stem cell growth factor designated“S1 factor,” erythropoietin and thrombopoietin. Examples of suitableimmunomodulator moieties include IL-2, IL-6, IL-10, IL-12, IL-18, IL-21,interferon-γ, TNF-α, and the like. Alternatively, subjects can receivenaked anti-CD20 antibodies and a separately administered cytokine, whichcan be administered before, concurrently or after administration of thenaked anti-CD20 antibodies. As discussed supra, the anti-CD20 antibodymay also be conjugated to the immunomodulator. The immunomodulator mayalso be conjugated to a hybrid antibody consisting of one or moreantibodies binding to different antigens.

Multimodal therapies of the present invention further includeimmunotherapy with naked anti-CD20 antibodies supplemented withadministration of anti-CD22, anti-CD19, anti-CD21, anti-CD74, anti-CD80,anti-CD23, anti-CD46 or HLA-DR (including the invariant chain)antibodies in the form of naked antibodies, fusion proteins, or asimmunoconjugates. The naked anti-CD20 antibodies or fragments thereofmay also be supplemented with naked antibodies against a MUC1 antigenthat is expressed on certain B-cells. These antibodies includepolyclonal, monoclonal, chimeric, human or humanized antibodies thatrecognize at least one epitope on these antigenic determinants.Anti-CD19 and anti-CD22 antibodies are known to those of skill in theart. See, for example, Ghetie et al., Cancer Res. 48:2610 (1988); Hekmanet al., Cancer Immunol. Immunother. 32:364 (1991); Longo, Curr. Opin.Oncol. 8:353 (1996) and U.S. Pat. Nos. 5,798,554 and 6,187,287,incorporated in their entirety by reference.

In another form of multimodal therapy, subjects receive naked anti-CD20antibodies, and/or immunoconjugates, in conjunction with standard cancerchemotherapy. For example, “CVB” (1.5 g/m² cyclophosphamide, 200-400mg/m² etoposide, and 150-200 mg/m² carmustine) is a regimen used totreat non-Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51: 18(1993). Other suitable combination chemotherapeutic regimens arewell-known to those of skill in the art. See, for example, Freedman etal., “Non-Hodgkin's Lymphomas,” in CANCER MEDICINE, VOLUME 2, 3rdEdition, Holland et al. (eds.), pages 2028-2068 (Lea & Febiger 1993). Asan illustration, first generation chemotherapeutic regimens fortreatment of intermediate-grade non-Hodgkin's lymphoma (NHL) includeC-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) andCHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). Auseful second generation chemotherapeutic regimen is m-BACOD(methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine,dexamethasone and leucovorin), while a suitable third generation regimenis MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine,prednisone, bleomycin and leucovorin). Additional useful drugs includephenyl butyrate and brostatin-1. In a preferred multimodal therapy, bothchemotherapeutic drugs and cytokines are co-administered with anantibody, immunoconjugate or fusion protein according to the presentinvention. The cytokines, chemotherapeutic drugs and antibody orimmunoconjugate can be administered in any order, or together.

In a preferred embodiment, NHL or the autoimmune disease is treated with4 weekly infusions of the humanized anti-CD20 antibody at a does of200-400 mg/m² weekly for 4 consecutive weeks (iv over 2-6 hours),repeated as needed over the next months/yrs. Preferably, the humanizedanti-CD-20 antibody is administered at a dose of 200-300 mg/m² onceevery other week or every third week, for 4 to 8 injections. Alsopreferred, NHL is treated with 4 weekly infusions as above, orinjections less frequently as above, but combined with epratuzumab(anti-CD22 humanized antibody) on the same days, at a dose of 360 mg/m²,given as iv infusion over 1 hour, either before, during or after theanti-CD20 monoclonal antibody infusion. Or, the antibodies used incombination therapy may also be infused in alternative sequences, suchthat they are alternated on different weeks, resulting in each beinggiven every other week for a total injection sequence for each of 4 to 8or more doses. These dosage schedules can then be repeated at differentintervals, such as every 3-6 months, depending on the patient's clinicalstatus and response to each therapy regimen. Still preferred, NHL istreated with 4 weekly infusions, or less frequent infusions, of theanti-CD20 antibody as above, combined with one or more injections ofCD22 MAb radiolabeled with a therapeutic isotope such as yttrium-90 (ata total dose of Y-90 between 5 and 35 mCi/meter-square as one or moreinjections over a period of weeks or months). U.S. Ser. No. 09/590,284(now U.S. Pat. No. 7,074,403—Goldenberg et al.) discloses immunotherapyof autoimmune disorders using an anti-CD22 antibody, which isincorporated herein by reference in its entirety.

In addition, a therapeutic composition of the present invention cancontain a mixture or hybrid molecules of monoclonal naked anti-CD20antibodies directed to different, non-blocking CD20 epitopes.Accordingly, the present invention contemplates therapeutic compositionscomprising a mixture of monoclonal anti-CD20 antibodies that bind atleast two CD20 epitopes. Additionally, the therapeutic compositiondescribed herein may contain a mixture of anti-CD20 antibodies withvarying CDR sequences.

Although naked anti-CD20 antibodies are the primary therapeuticcompositions for treatment of B cell lymphoma and autoimmune diseases,the efficacy of such antibody therapy can be enhanced by supplementingthe naked antibodies, with supplemental agents, such asimmunomodulators, like interferons, including IFNα, IFNβ and IFNγ,interleukins including IL-1, IL-2, IL-6, IL-12, IL-15, IL-18, IL-21, andcytokines including G-CSF and GM-CSF. Accordingly, the CD20 antibodiescan be combined not only with antibodies and cytokines, either asmixtures (given separately or in some predetermined dosing regiment) oras conjugates or fusion proteins to the anti-CD20 antibody, but also canbe given as a combination with drugs. For example, the anti-CD20antibody may be combined with CHOP as a 4-drug chemotherapy regimen.Additionally, a naked anti-CD20 antibody may be combined with a nakedanti-CD22 antibodies and CHOP or fludarabine as a drug combination forNHL therapy Immunotherapy of B-cell malignancies using an anti-CD22antibody is described in U.S. Pat. No. 6,183,744 (Goldenberg et al.) andU.S. Ser. No. 09/307,816 (now U.S. Pat. No. 6,306,393—Goldenberg etal.), which are incorporated herein by reference in their entirety. Thesupplemental therapeutic compositions can be administered before,concurrently or after administration of the anti-CD20 antibodies.

As discussed supra, the antibodies of the present invention can be usedfor treating B cell lymphoma and leukemia, and other B cell diseases ordisorders. For example, anti-CD20 antibodies can be used to treat B-cellrelated autoimmune diseases, including Class III autoimmune diseasessuch as immune-mediated thrombocytopenias, such as acute idiopathicthrombocytopenic purpura and chronic idiopathic thrombocytopenicpurpura, dermatomyositis, Sjögren's syndrome, multiple sclerosis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, rheumatoid arthritis, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, sarcoidosis,ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritisnodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitisubiterans, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis and fibrosing alveolitis.

Anti-CD20 antibodies may also induce apoptosis in cells expressing theCD20 antigen. Evidence of this induction is supported in the literature.For example, it was demonstrated that apoptosis could be induced usinglymphoid cells that have Fc-receptors reactive with the IgG1-Fc of CD20MAbs that crosslinked. See Shan et al., Cancer Immunol. Immunother.48(12):673-683 (2000). Further, it was reported that aggregates of achimeric CD20 MAb, i.e., homopolymers, induced apoptosis. See Ghetie etal., Blood 97(5): 1392-1398 (2000) and Ghetie et al., Proc. Natl. Acad.Sci. USA 94(14): 7509-7514 (1997).

Antibodies specific to the CD20 surface antigen of B cells can beinjected into a mammalian subject, which then bind to the CD20 cellsurface antigen of both normal and malignant B cells. A mammaliansubject includes humans and domestic animals, including pets, such asdogs and cats. The anti-CD20 mAbs of the present invention, i.e.,humanized, chimeric, human, caninized and felinized, and even murineanti-CD20 mAbs, can be used to treat the non-human mammalian subjectswhen there is a species crossreactivity for the CD20 antigen. SeeExamples 10 and 11, below. The murine mAbs, which are immunogenic inhumans, are usually less immunogenic in non-human mammalian subjects.The anti-CD20 antibody bound to the CD20 surface antigen leads to thedestruction and depletion of neoplastic B cells. Because both normal andmalignant B cells express the CD20 antigen, the anti-CD20 antibody willresult in B cell death. However, only normal B cells will repopulate andthe malignant B cells will be eradicated or significantly reduced.Additionally, chemical agents or radioactive labels having the potentialto destroy the tumor can be conjugated to the anti-CD20 antibody suchthat the agent is specifically targeted to the neoplastic B cells.

12. Expression Vectors

The DNA sequence encoding a humanized, chimeric or human anti-CD20 MAbcan be recombinantly engineered into a variety of known host vectorsthat provide for replication of the nucleic acid. These vectors can bedesigned, using known methods, to contain the elements necessary fordirecting transcription, translation, or both, of the nucleic acid in acell to which it is delivered. Known methodology can be used to generateexpression constructs the have a protein-coding sequence operably linkedwith appropriate transcriptional/translational control signals. Thesemethods include in vitro recombinant DNA techniques and synthetictechniques. For example, see Sambrook et al., 1989, MOLECULAR CLONING: ALABORATORY MANUAL, Cold Spring Harbor Laboratory (New York); Ausubel etal., 1997, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons(New York). Also provided for in this invention is the delivery of apolynucleotide not associated with a vector.

Vectors suitable for use in the instant invention can be viral ornon-viral. Particular examples of viral vectors include adenovirus, AAV,herpes simplex virus, lentivirus, and retrovirus vectors. An example ofa non-viral vector is a plasmid. In a preferred embodiment, the vectoris a plasmid.

An expression vector, as described herein, is a polynucleotidecomprising a gene that is expressed in a host cell. Typically, geneexpression is placed under the control of certain regulatory elements,including constitutive or inducible promoters, tissue-specificregulatory elements, and enhancers. Such a gene is said to be “operablylinked to” the regulatory elements.

Preferably, the expression vector of the instant invention comprises theDNA sequence encoding a humanized, chimeric or human anti-CD20 MAb,which includes both the heavy and the light chain variable and constantregions. However, two expression vectors may be used, with onecomprising the heavy chain variable and constant regions and the othercomprising the light chain variable and constant regions. Stillpreferred, the expression vector further comprises a promoter. Becauseany strong promoter can be used, a DNA sequence encoding a secretionsignal peptide, a genomic sequence encoding a human IgG1 heavy chainconstant region, an Ig enhancer element and at least one DNA sequenceencoding a selection marker.

Also contemplated herein is a method for expressing a humanizedanti-CD20 MAb, comprising (i) linearizing at least one expression vectorcomprising a DNA sequence encoding a humanized, chimeric, or humananti-CD20 MAb, (ii) transfecting mammalian cells with at least one ofsaid linearized vector, (iii) selecting transfected cells which expressa marker gene, and (iv) identifying the cells secreting the humanizedanti-CD20 MAb from the transfected cells.

13. Methods of Making Anti-CD20 Antibodies

In general, the Vκ (variable light chain) and V_(H) (variable heavychain) sequences for an anti-CD20 MAb can be obtained by a variety ofmolecular cloning procedures, such as RT-PCR, 5′-RACE, and cDNA libraryscreening. Specifically, the V genes of an anti-CD20 MAb can be clonedby PCR amplification from a cell that expresses a murine or chimericanti-CD20 MAb, sequenced. To confirm their authenticity, the clonedV_(L) and V_(H) genes can be expressed in cell culture as a chimeric Abas described by Orlandi et al., (Proc. Natl. Acad. Sci., USA, 86: 3833(1989)) which is incorporated by reference. Based on the V genesequences, a humanized anti-CD20 MAb can then be designed andconstructed as described by Leung et al. (Mol. Immunol., 32: 1413(1995)), which is incorporated by reference. cDNA can be prepared fromany known hybridoma line or transfected cell line producing a murine orchimeric anti-CD20 MAb by general molecular cloning techniques (Sambrooket al., Molecular Cloning, A laboratory manual, 2^(nd) Ed (1989)). TheVκ sequence for the MAb may be amplified using the primers VK1BACK andVK1FOR (Orlandi et al., 1989) or the extended primer set described byLeung et al. (BioTechniques, 15: 286 (1993)), which is incorporated byreference, while V_(H) sequences can be amplified using the primer pairVH1BACK/VH1FOR (Orlandi et al., 1989 above), or the primers annealing tothe constant region of murine IgG described by Leung et al. (Hybridoma,13:469 (1994)), which is incorporated by reference. The PCR reactionmixtures containing 10 μl of the first strand cDNA product, 10 μl of10×PCR buffer [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl₂, and0.01% (w/v) gelatin] (Perkin Elmer Cetus, Norwalk, Conn.), 250 μM ofeach dNTP, 200 nM of the primers, and 5 units of Taq DNA polymerase(Perkin Elmer Cetus) can be subjected to 30 cycles of PCR. Each PCRcycle preferably consists of denaturation at 94° C. for 1 min, annealingat 50° C. for 1.5 min, and polymerization at 72° C. for 1.5 min.Amplified Vκ and VH fragments can be purified on 2% agarose (BioRad,Richmond, Calif.). Similarly, the humanized V genes can be constructedby a combination of long oligonucleotide template syntheses and PCRamplification as described by Leung et al. (Mol. Immunol., 32: 1413(1995)). See Example 3 for a method for the synthesis of an oligo A andan oligo B on an automated RNA/DNA synthesizer (Applied Biosystems,foster City, Calif.) for use in constructing humanized V genes.

PCR products for Vκ can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites to facilitatein-frame ligation of the Vκ PCR products. PCR products for V_(H) can besubcloned into a similar staging vector, such as the pBluescript-basedVHpBS. Individual clones containing the respective PCR products may besequenced by, for example, the method of Sanger et al. (Proc. Natl.Acad. Sci., USA, 74: 5463 (1977)), which is incorporated by reference.

The DNA sequences described herein are to be taken as including allalleles, mutants and variants thereof, whether occurring naturally orinduced.

The expression cassettes containing the Vκ and VH, together with thepromoter and signal peptide sequences can be excised from VKpBR andVHpBS, respectively, by double restriction digestion as HindIII-BamHIfragments. The Vκ and VH expression cassettes can then be ligated intoappropriate expression vectors, such as pKh and pG1g, respectively(Leung et al., Hybridoma, 13:469 (1994)). The expression vectors can beco-transfected into an appropriate cell, e.g., myeloma Sp2/0-Ag14 (ATCC,VA), colonies selected for hygromycin resistance, and supernatant fluidsmonitored for production of a chimeric or humanized anti-CD20 MAb by,for example, an ELISA assay, as described below. Alternately, the Vκ andVH expression cassettes can be assembled in the modified stagingvectors, VKpBR2 and VHpBS2, excised as XbaI/BamHI and XhoI/BamHIfragments, respectively, and subcloned into a single expression vector,such as pdHL2, as described by Gilles et al. (J. Immunol. Methods125:191 (1989) and also shown in Losman et al., Cancer, 80:2660 (1997))for the expression in Sp2/0-Ag14 cells. Another vector that is useful inthe present invention is the GS vector, as described in Barnes et al.,Cytotechnology 32:109-123 (2000), which is preferably expressed in theNS0 cell line and CHO cells. Other appropriate mammalian expressionsystems are described in Werner et al., Arzneim.-Forsch./Drug Res.48(II), Nr. 8, 870-880 (1998).

Co-transfection and assay for antibody secreting clones by ELISA, can becarried out as follows. About 10 μg of VKpKh (light chain expressionvector) and 20 μg of VHpG1g (heavy chain expression vector) can be usedfor the transfection of 5×10⁶ SP2/0 myeloma cells by electroporation(BioRad, Richmond, Calif.) according to Co et al., J. Immunol., 148:1149 (1992) which is incorporated by reference. Following transfection,cells may be grown in 96-well microtiter plates in complete HSFM medium(Life Technologies, Inc., Grand Island, N.Y.) at 37° C., 5% CO₂. Theselection process can be initiated after two days by the addition ofhygromycin selection medium (Calbiochem, San Diego, Calif.) at a finalconcentration of 500 units/ml of hygromycin. Colonies typically emerge2-3 weeks post-electroporation. The cultures can then be expanded forfurther analysis.

Transfectoma clones that are positive for the secretion of chimeric orhumanized heavy chain can be identified by ELISA assay. Briefly,supernatant samples (˜100 μl) from transfectoma cultures are added intriplicate to ELISA microtiter plates precoated with goat anti-human(GAH)-IgG, F(ab′)₂ fragment-specific antibody (Jackson ImmunoResearch,West Grove, Pa.). Plates are incubated for 1 h at room temperature.Unbound proteins are removed by washing three times with wash buffer(PBS containing 0.05% polysorbate 20). Horseradish peroxidase (HRP)conjugated GAH-IgG, Fc fragment-specific antibodies (JacksonImmunoResearch) are added to the wells, (100 μl of antibody stockdiluted ×10⁴, supplemented with the unconjugated antibody to a finalconcentration of 1.0 μg/ml). Following an incubation of 1 h, the platesare washed, typically three times. A reaction solution, [100 μl,containing 167 μg of orthophenylene-diamine (OPD) (Sigma, St. Louis,Mo.), 0.025% hydrogen peroxide in PBS], is added to the wells. Color isallowed to develop in the dark for 30 minutes. The reaction is stoppedby the addition of 50 μl of 4 N HCl solution into each well beforemeasuring absorbance at 490 nm in an automated ELISA reader (Bio-Tekinstruments, Winooski, Vt.). Bound chimeric antibodies are thandetermined relative to an irrelevant chimeric antibody standard(obtainable from Scotgen, Ltd., Edinburg, Scotland).

Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2μ membrane. The filtered medium is passed througha protein A column (1×3 cm) at a flow rate of 1 ml/min. The resin isthen washed with about 10 column volumes of PBS and protein A-boundantibody is eluted from the column with 0.1 M glycine buffer (pH 3.5)containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3 M Tris (pH 8.6), and protein concentrationsdetermined from the absorbance at 280/260 nm. Peak fractions are pooled,dialyzed against PBS, and the antibody concentrated, for example, withthe Centricon 30 (Amicon, Beverly, Mass.). The antibody concentration isdetermined by ELISA, as before, and its concentration adjusted to about1 mg/ml using PBS. Sodium azide, 0.01% (w/v), is conveniently added tothe sample as preservative.

The following are the nucleotide sequences of the primers used toprepare the anti-CD20 antibodies:

hA20VKA (SEQ ID NO: 14) 5′-CATCTCTGAG CGCATCTGTT GGAGATAGGG TCACTATGACTTGTAGGGCC AGCTCAAGTG TAAGTTACAT CCACTGGTTCCAGCAGAAAC CAGGGAAAGC ACCTAAACCC TGGATTTATG-3′ hA20VKB (SEQ ID NO: 15)5′-GGTGTCCCTG TCCGATTCTC TGGCAGCGGA TCTGGGACAGATTACACTTT CACCATCAGC TCTCTTCAAC CAGAAGACATTGCAACATAT TATTGTCAGC AGTGGACTAG TAACCCACCC ACGTTCGGTG-3′hA20VKA-Backward (SEQ ID NO: 16)5′-CAGCTGACCC AGTCTCCATC ATCTCTGAGC GCATCTGTTG-3′ hA20VKA-Forward(SEQ ID NO: 17) 5′-AGGTTCGAAG TGGCATAAAT CCAGGGTTTA GGTGCT-3′hA20VKB Backward (SEQ ID NO: 18)5′-CACTTCGAAC CTGGCTTCTG GTGTCCCTGT CCGATTCTC-3′ hA20VKB Forward(SEQ ID NO: 19) 5′-ACGTTAGATC TCCAGCTTGG TCCCTCCACC GAACGTGGGT GGGTTA-3′hA20VHA (SEQ ID NO: 20) 5′-CTGAAGTCAA GAAACCTGGG TCATCGGTGA AGGTCTCCTGCAAGGCTTCT GGCTACACCT TTACTAGTTA CAATATGCACTGGGTCAAGC AGGCACCTGG ACAGGGTCTG GAATGGATTG G-3′ hA20VHB (SEQ ID NO: 21)5′-ATCAGAAGTT CAAGGGTAAA GCCACACTGA CTGCCGACGAATCCACCAAT ACAGCCTACA TGGAGCTGAG CAGCCTGAGGTCTGAGGACA CGGCATTTTA TTACTGTGCA AGATCGACTTACTACGGCGG TGACTGGTAC TTCGATGTCT G-3′ hA20VHA Backward (SEQ ID NO: 22)5′-CAGCTGCAGC AATCAGGGGC TGAAGTCAAG AAACCTGGG-3′ hA20VHA Forward(SEQ ID NO: 23) 5′-TTCCGGGATA AATAGCTCCA ATCCATTCCA GACCCTG-3′hA20VHB Backward (SEQ ID NO: 24)5′-ATCCCGGAAA TGGTGATACT TCCTACAATC AGAAGTTCAA GGGTAAAGCC A-3′hA20VHB Forward (SEQ ID NO: 25)5′-GGAGACGGTG ACCGTGGTGC CTTGGCCCCA GACATCGAAG TACCAG-3′ hA20VH2A(SEQ ID NO: 26) 5′-CTGAAGTCAA GAAACCTGGG TCATCAGTGA AGGTCTCCTGCAAGGCTTCT GGCTACACCT TTAGTAGTTA CAATATGCACTGGGTCAGAC AGGCACCTGG ACAGGGTCTG GAATGGATGG G-3′ hA20VH2B(SEQ ID NO: 27) 5′-ATCAGAAGTT CAAGGGTAGA GCCACAATAA CTGCCGACGAATCCACCAAT ACAGCCTACA TGGAGCTGAG CAGCCTGAGGTCTGAGGACA CGGCATTTTA TTTTTGTGCA AGATCGACTTACTACGGCGG TGACTGGTAC TTCGATGTCT G-3′ hA20VH2A Forward (SEQ ID NO: 28)5′-TTCCGGGATA AATAGCTCCC ATCCATTCCA GACCCTG-3′ hA20VH2B Backward(SEQ ID NO: 29) 5′-ATCCCGGAAA TGGTGATACT TCCTACAATC AGAAGTTCAAGGGTAGAGCC A-3′

The invention is further described by reference to the followingexamples, which are provided for illustration only. The invention is notlimited to the examples but rather includes all variations that areevident from the teachings provided herein.

EXAMPLES Example 1 Construction of a Humanized Anti-CD20 Antibody

The V_(H) and Vκ genes of A20, an anti-CD20 antibody, was obtained byRT-PCR using the primer pairs VH1BACK/VH1FOR and VK1BACK/VK1FOR,respectively Orlandi et al., (Proc. Natl. Acad. Sci., USA, 86: 3833(1989)). Multiple independent clones were sequenced to eliminatepossible errors resulting from the PCR reaction. The cloned murine V_(H)and Vκ sequences as the final PCR product were designated A20Vk (FIG.1A) and A20VH (FIG. 1B), respectively. A chimeric A20 (cA20) antibodywas constructed and expressed in Sp2/0 cell. The Vk and VH of sequencesof cA20 are shown in FIG. 2. The cA20 antibody bound to Raji cell andcompeted with radiolabeled A20 purified from the hybridoma cell culturesupernatant (FIG. 3). This result confirmed the authenticity of thecloned V genes.

A single light chain and two heavy chain variable region sequencesencoding the humanized anti-hCD20 (hA20) antibody were designed andconstructed. Human REI framework sequences were used for Vκ (FIG. 1A),and a combination of EU and NEWM framework sequences were used for V_(H)(FIG. 1B). There are a number of amino acid changes in each chainoutside of the CDR regions when compared to the starting human antibodyframeworks. The heavy chain of hA20, hA20V_(H)1, contains nine changes,while hA20V_(H)2 contains three changes from the human EU frameworks(FIG. 4A). hA20V_(H)2 is preferred because it contains more amino acidsfrom the human antibody framework region than hA20V_(H)1. The lightchain of hA20, hA20Vκ, contains seven amino acid changes from the REIframework (FIG. 4B).

Example 2 Method of hA20 Antibody Construction

Each variable chain was constructed in two parts, a 5′- and 3′-half,designated as “A” and “B” respectively. Each half was produced by PCRamplification of a single strand synthetic oligonucleotide template withtwo short flanking primers, using Taq polymerase. The amplifiedfragments were first cloned into the pCR4TA cloning vector fromInvitrogen (Carlsbad, Calif.) and subjected to DNA sequencing. Thetemplates and primer pairs are listed as follows:

Template Primers VKA VkA-Backward/VkA-Forward VKBVkB-Backward/VkB-Forward VH1A VHA-Backward/VH1A-Forward VH1BVH1B-Backward/VHB-Forward VH2A VHA-Backward/VH2A-Forward VH2BVH2B-Backward/VHB-Forward

Light Chain

For constructing the full-length DNA of the humanized Vκ sequence, oligohA20VKA (120 mer) and hA20VKB (130 mer) were synthesized on an automatedRNA/DNA synthesizer (Applied Biosystems). hA20VKA and B represent the nt26-145 and 166-195, respectively, of the hA20 Vκ (See FIG. 5A) OligohA20VKA and B were cleaved from the support and deprotected by treatmentwith concentrated ammonium hydroxide. After samples were vacuum-triedand resuspended in 100 μl of water, incomplete oligomers (less than100-mer) were removed by centrifugation through a ChormaSpin-100 column(Clontech, Palo Alto, Calif.). All flanking primers were preparedsimilarly, except ChromaSpin-30 columns were used to remove synthesisby-products. 1 μl of ChromaSpin column purified hA20VKA was PCRamplified in a reaction volume of 100 μl containing 10 μl of 10×PCRbuffer [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl₂, and 0.01%(w/v) gelatin] (Perkin Elmer Cetus, Norwalk, Conn.), 250 μM of eachdNTP, 200 nM of VkA-Backward and VkA-Forward, and 5 units of Taq DNApolymerase (Perkin Elmer Cetus). This reaction mixture was subjected to30 cycles of PCR reaction consisting of denaturation at 94° C. for 1min, annealing at 50° C. for 1.5 min, and polymerization at 72° C. for1.5 min hA20VKB was PCR-amplified by the primer pair VkB-Backward andVkB-Forward under similar condition. The amplified VKA and VKA fragmentswere purified on 2% agarose (BioRad, Richmond, Calif.). Uniquerestriction sites were designed at the ends of each fragment tofacilitate joining through DNA ligation. The amplified VKA fragmentcontained a PvuII restriction site, CAGCTG, at its 5′-end and a BstBIrestriction site, TTCGAA, at the 3′-end. The amplified VKB fragmentcontained a BstBI restriction site at its 5′-end and a BglII restrictionsite, AGATCT, at the 3′-end. Assembly of the full-length Vκ chain wasaccomplished by restriction enzyme digestion of each fragment with theappropriate 5′- and 3′-enzymes and ligation into the VKpBR2 vectorpreviously digested with PvuII and BclI (BclI digested end is compatiblewith that of BglII). The resulting ligated product contains the Afragment ligated to the PvuII site, the B fragment ligated to the BclIsite, and the A and B fragments joined together at the BstBI site (FIG.5A). VKpBR2 is a modified staging vector of VKpBR (Leung et al.,Hybridoma, 13:469 (1994)), into which a XbaI restriction site wasintroduced at 14 bases upstream of the translation initiation codon.Upon confirmation of a correct open reading frame by DNA sequencing, theintact chain was removed from VKpBR2 as a XbaI to BamHI fragment andligated into the pdHL2 expression vector. The vector containing only Vκsequence was designated as hA20VκpdHL2. pdHL2 contains the expressioncassettes for both human IgG1 C1, C2, C3, and hinge regions (FIG. 7A)and the human kappa chain Ck (FIG. 7B) under the control of IgH enhancerand MT_(I) promoter, as well as a mouse dhfr gene, controlled by a weakSV40 promotor, as a marker for selection of transfectants andco-amplification of the trans-genes (Gillies et al., J. Immunol. Methods125:191 (1989); Losman et al., Cancer 80:2660 (1997)). By replacing theVκ and VH segments of pdHL2, different chimeric or humanized Abs can beexpressed.

Heavy Chain

For the construction of hA20VH1, oligo VH1A (121 mer) and VH1B (151mer), representing the nt 23-143 and 179-329, respectively, (See FIG.5B) were synthesized as described above. Similarly, for hA20VH2, oligoVH2A and VH2B were prepared. These oligos were PCR-amplified by theirrespective primer pairs as listed in Example 2. The same constructionmethod as done for V was carried out for both types of VH, with thefollowing modifications: the 5′-end restriction site of the A fragmentswas PstI (CTGCAG) and the 3′-end restriction site of B fragments wasBstElI (GGTCACC). These fragments were joined together upon ligationinto the VHpBS2 vector at a common NciI site (CCCGG), resulting infull-length VH sequences (FIGS. 5B and 5C) and confirmed by DNAsequencing. VHpBS2 is a modified staging vector of VHpBS (Leung et al.,Hybridoma, 13:469 (1994)), into which a Xho1 restriction site wasintroduced at 16 bases upstream of the translation initiation codon. Theassembled Vii genes were subcloned as XhoI-BamHI restriction fragmentsinto the expression vector containing the V sequence, hA20V pdHL2. Sincethe heavy chain region of pdHL2 lacks a BamH1 restriction site, thisligation required use of the HNB linker to provide a bridge between theBamH1 site of the variable chain and the Hindlll site present in thepdHL2 vector. The resulting expression vectors were designated as hA20-1pdHL2 and hA20-2 pdHL2.

HNB linker (SEQ ID NO: 30) 5′-AGCTTGCGGCCGC-3′ (SEQ ID NO: 31)3′-ACGCCGGCGCTAG-5′

Example 3 Transfection and Expression of hA20 Antibodies

Approximately 30 μg of the expression vectors for hA20 were linearizedby digestion with SalI and transfected into Sp2/0-Ag14 cells byelectroporation (450V and 25 μF). The transfected cells were plated into96-well plates for 2 days and then selected for drug-resistance byadding MTX into the medium at a final concentration of 0.025 μM.MTX-resistant colonies emerged in the wells 2-3 weeks. Supernatants fromcolonies surviving selection were screened for human Ab secretion byELISA assay. Briefly, 100 μl supernatants were added into the wells of amicrotiter plate precoated with GAH-IgG, F(ab′)₂ fragment-specific Aband incubated for 1 h at room temperature. Unbound proteins were removedby washing three times with wash buffer (PBS containing 0.05%polysorbate 20). HRP-conjugated GAH-IgG, Fc fragment-specific Ab wasadded to the wells. Following an incubation of 1 h, the plate waswashed. The bound HRP-conjugated Ab was revealed by reading A490 nmafter the addition of a substrate solution containing 4 mM OPD and 0.04%H₂O₂. Positive cell clones were expanded and hA20-1 and hA20-2 werepurified from cell culture supernatant by affinity chromatography on aProtein A column.

Example 4 Binding Activity of Anti-CD20 Antibodies

A competition cell-binding assay was carried out to assess theimmunoreactivity of hA20 relative to the parent cA20 and the anti-CD20Ab c2B8. A constant amount of ¹²⁵I-labeled murine A20 or c2B8 (100,000cpm, ˜10 μCi/μg) was incubated with Raji cell in the presence of varyingconcentrations (0.2-700 nM) of hA20-1, -2, murine A20, cA20, or c2B8 at4° C. for 1-2 h. Unbound Abs were removed by washing the cells in PBS.The radioactivity associated with cells was determined after washing. Asshown in FIG. 6, both humanized A20 mAbs, hA20-1 and hA20-2, exhibitedcomparable binding activities as A20, the murine anti-CD20 MAb, cA20,and c2B8, a chimeric anti-CD20 MAb, when competing with binding of¹²⁵I-A20 or ¹²⁵I-c2B8 to Raji cells.

By direct binding of radiolabeled Mabs to Raji cells and Scatchard plotanlaysis, the dissociation constants were measured to be 2.9 and 4.2 nmfor cA20 and hA20, respectively, in comparison to 3.9 nM for C2B8. Invitro crosslinking experiments, using a goat anti-human IgG, Fc fragmentspecific antibody to complex with the antibodies showed similar killingof Raji NHL cells between cA20 and hA20, as well as C2B8.

Example 5 Treatment of a Patient with Relapsed Intermediate-GradeNon-Hodgkin's Lymphoma

A patient with intermediate grade non-Hodgkin's lymphoma has failedprior aggressive chemotherapy, consisting of CHOP×6, which led to acomplete remission for four months, another course of CHOP×6, resultingin progression, D-MOPP×2, resulting in stable disease for three months,and CVB with peripheral stem cell transplantation, which led to apartial remission for five months. The patient presents with recurrentlymphoma in a neck lymph node, measurable by computerized tomography andpalpation.

The patient is infused within 3 hrs with 450 mg of humanized CD20monoclonal antibody A20 on days 0, 14, 28, and 42 with no seriousadverse effects noted either during or immediately after the infusions.Eight weeks later, palpation of the neck node enlargement shows ameasurable decrease of about 50%. Follow-up measurements made at twentyweeks post therapy show no evidence of the disease in the neck, andnowhere else, as confirmed by computed tomography studies of the body.Since new disease is not detected elsewhere, the patient is consideredto be in complete remission. Follow-up studies every 10-12 weeksconfirms a complete remission for at least ten months post therapy.

Example 6 Treatment of a Patient with Chronic IdiopathicThrombocytopenia Purpura

A 45-year-old female with chronic idiopathic thrombocytopenia purpurahas been treated with prednisone, gamma globulins, and high dosedexamethasone, but the disease progresses. She undergoes splenectomy,which fails to stabilize the disease. Her platelet count falls to lessthan 30,000/microliter, and hemorrhagic events increase in frequency.The patient is then treated with the humanized CD20 A20 MAb, 500 mgintravenously on the first week, followed by a dose of 250 mg given onceevery other week for a total of 4 injections. Ten weeks after the lastdose of A20 a marked increase in platelet number is observed, to150,000/microliter, and the hemorrhagic events disappear. Five monthsafter the last antibody infusion the disease is in remission.

Example 7 Treatment of a Patient with Progressive Rheumatoid Arthritis

A 70 year old female, with severe progressive rheumatoid arthritis ofthe finger joints, wrists, and elbows, has failed therapy withmethotrexate, and obtains only minor relief when placed on Enbreltherapy. The patient is then treated with A20 humanized CD20 MAb, 300 mgintravenously each week, for a period of four weeks. After 3 months, a40% improvement in measures of disease activity is observed, which ismaintained for 5 months. The patient is again treated with A20, at thesame dose and frequency. The patient continues to improve, and 6 monthsafter the second A20 MAb therapy, a 60% improvement is observed. Nohuman anti-A20 antibodies are observed at any time during, or after theA20 therapy. Although normal B-cells are depleted from the blood, noinfectious complications, or other drug-related severe toxicity isobserved.

Example 8 Treatment of a Patient with Myasthenia Gravis

A 65 year old male has failed all conventional therapy for myastheniagravis, and is admitted to a neurological intensive therapy unit. Thepatient was stabilized by plasma exchange, and given intravenousimmunoglobulin to reduce the titer of antiacetylcholine receptorantibody. The patient remained bedridden, and was then treated with A20humanized CD20 MAb, 400 mg intravenously once every other week, for aperiod of ten weeks. One week after the last dose of A20, no bloodB-cells were detectable, and a significant drop in the titer of theanti-acetylcholine antibody was observed. Four months after the last A20MAb dose the patient was mobile, and was released from the hospital.

Example 9 Treatment of a Dog with Aggressive Non-Hodgkin's B-CellLymphoma in Lymph Nodes and Bone Marrow

A 65-pound, 7-year old male Golden Retriever is diagnosed with diffuselarge cell aggressive lymphoma. The dog is placed on combinationchemotherapy with vincristine, cyclophosphamide, prednisolone, anddoxorubicin, with good response. However, the dog subsequently ischaracterized as having progressive lymphadenopathy, and seven monthsafter this is found to have extensive lymphoma infiltration of bonemarrow, extensive lymphoadenopathy of neck, chest, abdomen, pelvis, andhepatosplenomegaly.

The dog is given therapy with 1F5 chimeric monoclonal antibody. The dogis infused intravenously with 120 mg of 1F5 antibody, and the treatmentis repeated weekly for 4 weeks following this initial treatment. Fourmonths after the final dose of 1F5, a computerized tomography scan ofthe patient shows no evidence of lymphoma, and all signs and symptoms ofthe disease were not evident.

Example 10 Treatment of a Dog with Relapsed Intermediate-GradeNon-Hodgkin's Lymphoma

A 78-pound, 9-year old, German Shepherd dog with intermediate gradenon-Hodgkin's lymphoma receives chemotherapy, which initially leads to acomplete remission for five months, followed by another course ofchemotherapy which results in stable disease for six months. The dogthen presents with recurrent lymphoma in the chest and in a neck lymphnode, both measurable by computerized tomography and palpation,respectively.

The patient is infused with a ⁹⁰Y-labeled immunoconjugate of L243(HLA-DR) monoclonal antibody weekly for two weeks, at a radiation doseof 8 mCi in 50 mg of antibody protein, in combination with the A20humanized CD20 antibody at a dose of 100 mg per each weekly infusion.Three weeks later, palpation of the neck node enlargement shows ameasurable decrease, while a repeat computerized tomography scan of thechest shows a marked reduction in tumor. Follow-up measurements made atten weeks post therapy show evidence of the disease in the neck or thechest being reduced by a about 60 percent. Since new disease is notdetected elsewhere, the patient is considered to be in partialremission. Follow-up studies every 10-12 weeks confirms a partialremission for at least 7 months post therapy.

Example 11 Treatment of a Cat with Relapsed Lymphoma

A 10-pound, 12-year-old, domestic short hair presents with enlargementof a single submandibular lymph node. After excision, there isrecurrence of the lesion at 6 months. The lesion is again excised, butthen reappears 6 months later. The cat is then given weekly treatmentsfor 4 weeks with an ¹³¹I-labeled immunoconjugate of anti-CD20 B1monoclonal antibody, at a radiation dose of 15 mCi in 45 mg antibodyprotein. The treatment is repeated 3 months later. When examined 3months after the last treatment, a marked decrease can be palpated. Norecurrence of the disease is observed for over one year.

Example 12 Evaluation of Chimeric and Humanized Anti-CD20 Mabs in HumanNHL Cells in Culture or Xenografted in SCID Mice

The properties of a chimeric (cA20) and humanized (hA20) CD20 antibodywas assessed in NHL cell lines. The results demonstrate that cA20 andhA20 behave similarly to Rituximab, staining more than 99% of Raji,Ramos, RL, Daudi and Su-DHL-6 cells and reacting with approximately 5%of lymphocytes (expected % B-cells). In all B-cell lines, specificgrowth inhibition was seen with the Mabs, but the level of inhibitionvaried between the cell lines, with Su-DHL being the most sensitive. Inthe absence of cross-linking, murine anti-CD20, cA20, hA20 and rituximaball yielded between 77 and 90% inhibition. With cross-linking,inhibition of proliferataion ranged from 94-98%. Rituximab, cA20, andhA20 were also similar in their ability to induce apoptosis in Rajicells in the presence of a cross-linking second monoclonal antibody.

Also, SCID mice were injected intravenously with 2.5×10⁶ Raji cells onday 0. Injections of murine, chimeric and humanized anti-CD20antibodies, and the cA20 F(ab′)₂ fragment were initiated on day-1 with100

g/injection of intact antibody, or 67

g/injection F(ab′)₂ fragment, five times per week for two weeks, thetwice weekly for three weeks. In one study, control mice died ofdisseminated disease with a median survival time of 15 days post tumorinoculation, but median survival was extended to 38 days for cA20, 22.5days for hA20, and 21 days for murine anti-CD20 treated mice (allstatistically significant by log-rank analysis (p<0.005)). In anotherstudy, control mice died of disseminated disease manifested with CNSparalysis with a median survival time of 16.5 days post tumorinoculation, but median survival was extended to 105 days for cA20, 70days for hA20, and 98 days for rituximab treated mice (all statisticallysignificant extensions by log-rank analysis (p<0.0001), FIG. 11).

Example 13 Competitive Cell Surface Binding Assay

Ag-binding specificity and affinity studies of humanized anti-CD20 Abs(cA20, hA20, and c1F5), purified by affinity chromatography on a ProteinA column) were evaluated by a cell surface competitive binding assaywith murine 2B8 and rituximab (IDEC Pharmaceuticals Corp., San Diego,Calif.) (FIG. 8). Briefly, a constant amount (100,000 cpm, ˜10 ìCi/ìg)of ¹²⁵I-labeled (A) m2B8 or (B) rituximab was incubated with Raji cellsin the presence of varying concentrations (0.2-700 nM) of competing Abs(cA20, hA20, m2B8, c1F5, or rituximab) at 4° C. for 1-2 h. Unbound Abswere removed by washing the cells with PBS. Radioactivity associatedwith the cells was determined after washing. FIG. 8 (A) is a comparisonof the binding activities of cA20 (square), hA20-1 (triangle) and hA20-1(circle) with that of m2B8 (diamond); FIG. 8 (B) Compares the bindingactivities of cA20 (square), c1F5 (triangle) and rituximab (diamond).

In another study, the binding activities of hA20 with other anti-CD20Abs, rituximab and murine B1 were compared by a cell surface competitivebinding assay (FIG. 9). Briefly, a constant amount (100,000 cpm, ˜10ìCi/ìg) of ¹²⁵I-labeled rituximab was incubated with Raji cells in thepresence of varying concentrations (0.2-700 nM) of competing Abs, hA20(triangle), mB1 (Downward triangle) or rituximab (square) at 4° C. for1-2 h. Unbound Abs were removed by washing the cells with PBS.Radioactivity associated with the cells was determined after washing.The IC₅₀ values for these three Abs were calculated to be 6.8, 34, and5, respectively.

Example 14 Cytotoxic Effect of Crosslinked hA20 and Other CD20 Abs onCultured Lymphoma Cells

Raji cells were treated with various CD20 Abs in the presence of acrosslinker (an anti-human IgG, Fc fragment specific antibody) tocomplex the CD20 antibodies (FIG. 10). A final concentration of 5 ìg/mlof hA20, cA20, rituximab, or a positive control Ab, hLL1, was incubatedwith Raji cells, with 20 ìg/ml of the crosslinker (red), without thecrosslinker (orange), or with an anti-mouse IgG, Fc fragment specificantibody (blue) for 48 h. Total cell and viable cell populations weremeasured by (A) trypan blue staining and cell counting or (B) MTT assay(B). The data show a similar effect of hA20 and rituximab on Raji NHLcell survival, and that the cytotoxic effect is dependent on thespecific crosslinking of the antibodies.

Example 15 In Vivo Therapy with hA20 and hLL2

Raji cells wer administered i.v. to 60 SCID mice, at 2.5×10⁶ cells/100μl/mouse (FIG. 12). MAbs were administered i.p. on days 1 to 11,followed by MAb injections twice per week, for approximately 3 weeks.The body weight of the animals was measured weekly until the study wasterminated. The animals were examined daily for paralysis of the hindlegs. When paralysis occurred, the animals were sacrificed andnecropsied for visual inspection of disseminated tumor nodules(specifically in lungs, kidneys, and ovaries). Control mice treated witha control humanized IgG1 Ab, hMN-14 (an anti-CEA antibody), died ofdisseminated disease manifested with CNS paralysis. The median survivaltime was 13 days post tumor i.v. inoculation. Median survival in thegroup treated with hA20 was extended to about 25 days. This valuerepresents median survival increase of approximately 2 fold for hA20.Although the group treated with hLL2 alone showed the same mediansurvival time compared to the control mice, treatment with combinationof hA20 and hLL2 increased the median survival time of the mice toapproximately 30 days.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention, which isdefined by the following claims.

All of the publications and patent applications and patents cited inthis specification are herein incorporated in their entirety byreference.

1. An isolated nucleic acid encoding a chimeric or humanized monoclonalantibody or antigen-binding fragment thereof that binds to CD20, saidantibody having a light chain variable region CDR1 comprising thesequence of RASSSVSYIH (SEQ ID NO:1); CDR2 comprising the sequence ofATSNLAS (SEQ ID NO:4); and CDR3 comprising the sequence of QQWTSNPPT(SEQ ID NO:4); and a heavy chain variable region CDR1 comprising thesequence of SYNMH (SEQ ID NO:8); CDR2 comprising the sequence ofAIYPGNGDTSYNQKFKG (SEQ ID NO:9); and CDR3 comprising the sequence ofSTYYGGDWYFDV (SEQ ID NO:10) or the sequence of VVYYSNSYWYFDV (SEQ IDNO:13).
 2. The isolated nucleic acid according to claim 1, wherein saidantibody is a chimeric monoclonal antibody.
 3. The isolated nucleic acidaccording to claim 1, wherein said antibody is a humanized monoclonalantibody.
 4. The isolated nucleic acid according to claim 1, wherein theheavy chain variable region CDR3 comprises the sequence of STYYGGDWYFDV(SEQ ID NO:10).
 5. The isolated nucleic acid according to claim 1,wherein the heavy chain variable region CDR3 comprises the sequence ofVVYYSNSYWYFDV (SEQ ID NO:13).
 6. The isolated nucleic acid according toclaim 1, wherein said antibody or fragment thereof is a fusion protein.7. The isolated nucleic acid according to claim 6, wherein said fusionprotein comprises a second monoclonal antibody or antigen-bindingfragment thereof.
 8. The isolated nucleic acid according to claim 7,wherein said second monoclonal antibody or fragment thereof is ananti-CD20 antibody or fragment thereof.
 9. The isolated nucleic acidaccording to claim 7, wherein said second monoclonal antibody orfragment thereof binds to an antigen selected from the group consistingof CD4, CD5, CD8, CD14, CD15, CD19, CD21, CD22, CD23, CD25, CD33, CD37,CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, B7, MUC1, MUC2,MUC3, MUC4, Ia, HM1.24, HLA-DR, tenascin, VEGF, PlGF, and an oncogeneproduct.
 10. An expression vector comprising the isolated nucleic acidof claim
 1. 11. The expression vector according to claim 10, wherein theexpression vector is a pdHL2 vector.
 12. A host cell comprising theexpression vector of claim
 10. 13. The host cell according to claim 12,wherein the host cell is a bacterial cell, a eukaryotic cell or amammalian cell.
 14. A method for production of a chimeric or humanizedmonoclonal antibody or antigen-binding fragment thereof that binds toCD20 comprising: a. transfecting a host cell with an expression vectoraccording to claim 10; and b. culturing the host cell in culture mediumso that it produces the chimeric or humanized monoclonal anti-CD20antibody or fragment thereof or fusion protein.
 15. The method of claim14, wherein the host cell secretes the anti-CD20 antibody or fragmentthereof or fusion protein into the culture medium.
 16. The method ofclaim 15, further comprising purifying the anti-CD20 antibody orfragment thereof or fusion protein from the culture medium.
 17. Themethod of claim 15, wherein the host cell is a bacterial host cell, aeukaryotic host cell or a mammalian host cell.
 18. A pharmaceuticalcomposition comprising a chimeric or humanized monoclonal antibody orantigen-binding fragment thereof that binds to CD20, said antibodyhaving a light chain variable region CDR1 comprising the sequence ofRASSSVSYIH (SEQ ID NO:1); CDR2 comprising the sequence of ATSNLAS (SEQID NO:4); and CDR3 comprising the sequence of QQWTSNPPT (SEQ ID NO:4);and a heavy chain variable region CDR1 comprising the sequence of SYNMH(SEQ ID NO:8); CDR2 comprising the sequence of AIYPGNGDTSYNQKFKG (SEQ IDNO:9); and CDR3 comprising the sequence of STYYGGDWYFDV (SEQ ID NO:10)or the sequence of VVYYSNSYWYFDV (SEQ ID NO:13).
 19. The pharmaceuticalcomposition according to claim 18, wherein said antibody is a chimericmonoclonal antibody.
 20. The pharmaceutical composition according toclaim 18, wherein said antibody is a humanized monoclonal antibody. 21.The pharmaceutical composition according to claim 18, wherein the heavychain variable region CDR3 comprises the sequence of STYYGGDWYFDV (SEQID NO:10).
 22. The pharmaceutical composition according to claim 18,wherein the heavy chain variable region CDR3 comprises the sequence ofVVYYSNSYWYFDV (SEQ ID NO:13).
 23. The pharmaceutical compositionaccording to claim 18, wherein said antibody or fragment thereof is afusion protein.
 24. The pharmaceutical composition according to claim23, wherein said fusion protein comprises a second monoclonal antibodyor antigen-binding fragment thereof.
 25. The pharmaceutical compositionaccording to claim 24, wherein said second monoclonal antibody orfragment thereof is an anti-CD20 antibody or fragment thereof.
 26. Thepharmaceutical composition according to claim 24, wherein said secondmonoclonal antibody or fragment thereof binds to an antigen selectedfrom the group consisting of CD4, CD5, CD8, CD14, CD15, CD19, CD21,CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74,CD80, CD126, B7, MUC1, MUC2, MUC3, MUC4, Ia, HM1.24, HLA-DR, tenascin,VEGF, PlGF, and an oncogene product.
 27. A kit comprising a chimeric orhumanized monoclonal antibody or antigen-binding fragment thereof thatbinds to CD20, said antibody having a light chain variable region CDR1comprising the sequence of RASSSVSYIH (SEQ ID NO:1); CDR2 comprising thesequence of ATSNLAS (SEQ ID NO:4); and CDR3 comprising the sequence ofQQWTSNPPT (SEQ ID NO:4); and a heavy chain variable region CDR1comprising the sequence of SYNMH (SEQ ID NO:8); CDR2 comprising thesequence of AIYPGNGDTSYNQKFKG (SEQ ID NO:9); and CDR3 comprising thesequence of STYYGGDWYFDV (SEQ ID NO:10) or the sequence of VVYYSNSYWYFDV(SEQ ID NO:13).
 28. The kit according to claim 27, wherein said antibodyis a chimeric monoclonal antibody.
 29. The kit according to claim 27,wherein said antibody is a humanized monoclonal antibody.
 30. The kitaccording to claim 27, wherein the heavy chain variable region CDR3comprises the sequence of STYYGGDWYFDV (SEQ ID NO:10).
 31. The kitaccording to claim 27, wherein the heavy chain variable region CDR3comprises the sequence of VVYYSNSYWYFDV (SEQ ID NO:13).
 32. The kitaccording to claim 27, wherein said antibody or fragment thereof is afusion protein.
 33. The kit according to claim 32, wherein said fusionprotein comprises a second monoclonal antibody or antigen-bindingfragment thereof.
 34. The kit according to claim 33, wherein said secondmonoclonal antibody or fragment thereof is an anti-CD20 antibody orfragment thereof.
 35. The kit according to claim 33, wherein said secondmonoclonal antibody or fragment thereof binds to an antigen selectedfrom the group consisting of CD4, CD5, CD8, CD14, CD15, CD19, CD21,CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74,CD80, CD126, B7, MUC1, MUC2, MUC3, MUC4, Ia, HM1.24, HLA-DR, tenascin,VEGF, PlGF, and an oncogene product.
 36. An immunoconjugate comprising:a. a chimeric or humanized monoclonal antibody or antigen-bindingfragment thereof that binds to CD20, said antibody having a light chainvariable region CDR1 comprising the sequence of RASSSVSYIH (SEQ IDNO:1); CDR2 comprising the sequence of ATSNLAS (SEQ ID NO:4); and CDR3comprising the sequence of QQWTSNPPT (SEQ ID NO:4); and a heavy chainvariable region CDR1 comprising the sequence of SYNMH (SEQ ID NO:8);CDR2 comprising the sequence of AIYPGNGDTSYNQKFKG (SEQ ID NO:9); andCDR3 comprising the sequence of STYYGGDWYFDV (SEQ ID NO:10) or thesequence of VVYYSNSYWYFDV (SEQ ID NO:13); and b. at least onetherapeutic and/or diagnostic agent attached to the chimeric orhumanized monoclonal antibody or fragment thereof.
 37. Theimmunoconjugate according to claim 36, wherein said chimeric orhumanized anti-CD20 antibody or fragment thereof is a fusion protein.38. The immunoconjugate according to claim 36, wherein said therapeuticagent is selected from the group consisting of a radioisotope, acytotoxic agent, a drug, a toxin, a second antibody, a secondantigen-binding antibody fragment and an immunomodulator.
 39. Theimmunoconjugate according to claim 38, wherein said immunomodulator isselected from the group consisting of a cytokine, an interferon, a stemcell growth factor, thrombopoietin, a lymphotoxin and a colonystimulating factor.
 40. A method of treating an autoimmune disease or aB-cell lymphoma or leukemia in a subject comprising administering tosaid subject a pharmaceutical composition according to claim
 18. 41. Amethod of treating an autoimmune disease or a B-cell lymphoma orleukemia in a subject comprising administering to said subject animmunoconjugate according to claim 39.